Antimicrobial coatings and methods of making and using thereof

ABSTRACT

Disclosed are antimicrobial articles and surfaces, as well as methods of making such articles and surfaces.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 16/305,534,filed Nov. 29, 2018, which claims benefit of U.S. ProvisionalApplication No. 62/343,317, filed May 31, 2016, and U.S. ProvisionalApplication No. 62/393,684, filed Sep. 13, 2016, each of which arehereby incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under Grant No. CHE1011796 awarded by the National Science Foundation. The Government hascertain rights in the invention.

BACKGROUND

Infection acquired from health care environments is one of the leadingmajor medical complications in the present world. Studies have shownthat almost 6% of patients admitted to hospitals acquire infections andthe number of such cases is increasing. According to reports by the U.S.Centers for Disease Control and Prevention, hospital acquired infectionsaccount for more than 2 million cases leading to 99,000 deaths annually.

The most common hospital acquired infections include urinary tractinfections, surgical wound infections, and those associated withintravascular cannulas. The mode of transmission of these infections ismostly by physical contact with infected medical devices. Staphylococcusaureus, Pseudomonas aeruginosa, and Escherichia coli are the most commonbacterial isolates that give rise to these infectious diseases. It hasbeen observed that most of the bacterial strains develop resistance toantibiotics over a period of time. In the hospital environment, over 50%of Staphylococcus aureus have developed resistance to methicillin, whichultimately leads to surgical wound infection and catheter relatedsepsis. Some of the emerging antibiotic resistant pathogens includevancomycin resistant Enterococci, vancomycin intermediateStaphylococcus, and multiple antibiotic resistant Gram negativeorganisms like Acinetobacter, Enterobacter, and mycobacterium.

Antimicrobial surfaces employed, for example, on the surface of medicaldevices, offer promise in helping curb the spread of infections. Towardsthis end, a wide range of antimicrobial agents have been applied tosurfaces: antibiotics including chlorhexidine, rifampin and monocyclineand others, silver/silver ions/silver compounds, hydantoin (also knownas halamine) compounds, furanone compounds, and quaternary ammonium orphosphonium polymers. There have been a smaller number ofnon-permanently cationic antimicrobial polymeric materials prepared foruse on surfaces, generally incorporating benzoic acid derivatives.

The various agents are most often physically applied to the surface,physically impregnated into the bulk of the material, or physicallyincorporated into a coating that is then applied to the surface for“controlled release”. In all these approaches the antimicrobial agentleaches from the surface, leading to two key problems: a limited time ofeffectiveness; and environmental, health and safety concerns, such asthe promotion of drug resistant microbes.

Non-leaching antimicrobial surfaces have been created by covalentlygrafting an antimicrobial polymer to the surface, atom transfer radicalpolymerization of an antimicrobial polymer directly from an initiatingsurface, and covalent attachment of an agent to a polymer chain. In thelatter case, any attachment scheme must not obscure the active moiety ofthe molecule. Also, particular care must be taken to ensure that theagent is actually covalently bound and is not just physicallyincorporated and that it is not releasing from the surface, which leadsto the same issues discussed above for leaching antimicrobial agents.

While non-leaching systems address some of the shortcomings associatedwith the “controlled release” architectures described above, manyexisting non-leaching systems are not compatible commercially-viablemanufacturing methods such as molding, extrusion, and otherthermoplastic methods of ‘conversion’ or solvent-based processing.Further, many existing non-leaching coatings unacceptably alter thephysiochemical and mechanical properties of the substrate to which theyare applied.

SUMMARY

Provided herein are processes for rendering a polyvinyl chloride (PVC)surface antimicrobial (e.g., antibacterial and/or antifungal). Theprocesses generally involve functionalization of the PVC surface toconvert chloride residues on the PVC surface to functional groups (e.g.,an alkyne or an azide) that can participate in a click reaction (e.g., a1,3-dipolar cycloaddition reaction). Subsequently, click chemistry canbe used to covalently tether one or more antimicrobial agents to thesurface. The resulting surfaces can exhibit antimicrobial activity(e.g., antibacterial activity, antifungal activity, or a combinationthereof).

Importantly, these processes can employ mild reaction conditions thatpermit covalent attachment of the antimicrobial agents to a PVC surfacewithout substantially altering the physiochemical and mechanicalproperties of the article comprising the PVC surface. As a consequence,the processes described herein can be used to impart antimicrobialactivity to a surface of a formed PVC article (e.g., an extruded articlesuch as PVC tubing) without unacceptably compromising the physiochemicaland mechanical properties of the article (e.g., without damaging the PVCtubing).

In some embodiments, the process for rendering a polyvinyl chloridesurface antimicrobial can comprise (a) contacting the polyvinyl chloridewith an azidation reagent in the presence of a phase transfer catalystto form an azide-substituted polyvinyl chloride; and (b) contacting theazide-substituted polyvinyl chloride with an antimicrobial agentcomprising an alkyne moiety under conditions effective to covalentlybond the antimicrobial agent to the polyvinyl chloride surface.

In other embodiments, the process for rendering a polyvinyl chloridesurface antimicrobial can comprise (a) contacting the polyvinyl chloridewith a cyanation reagent in the presence of a phase transfer catalyst toform a cyano-substituted polyvinyl chloride; and (b) covalently bondingan antimicrobial agent to the cyano-substituted polyvinyl chloride. Alsoprovided are antimicrobial articles. The antimicrobial articles cancomprise a surface (e.g., a surface of a substrate), and a polycationicpolymer derived from the condensation of a cyclic bis-electrophile and apolynucleophilic monomer immobilized on the surface. The cyclicbis-electrophile can be defined by Formula II below

where each dotted line represents a cyclic moiety; LG represents aleaving group; Z represents S, Se, or NR³; and R³ represents alkyl,alkenyl, alkynyl, alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl,cycloalkyl alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl,peptidyl, polyamino, or polyalkyleneoxy. In some embodiments, the cyclicbis-electrophile can be defined by Formula IIA, Formula IIB, or FormulaIIC below

wherein LG and Z are as defined above with respect to Formula II. Thepolynucleophilic monomer can be defined by Formula III below

wherein A represents a heterocyclic ring comprising a tertiary nitrogenatom; Y is absent or represents a linking group; and R¹ is absent, orrepresents halogen, hydroxy, amino, cyano, azido, hydrazone, alkyl,alkenyl, alkynyl, alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl,cycloalkyl alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, dialkylamino,alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,peptidyl, polyamino, or polyalkyleneoxy.

Also provided are methods for controlling biofilm formation on asubstrate. Methods can comprise covalently bonding a polycationicpolymer to the substrate in an amount effective to inhibit biofilmformation. The polycationic polymer can be derived from the condensationof a cyclic bis-electrophile and a polynucleophilic monomer immobilizedon the surface. The cyclic bis-electrophile can be defined by Formula IIbelow

where each dotted line represents a cyclic moiety; LG represents aleaving group; Z represents S, Se, or NR³; and R³ represents alkyl,alkenyl, alkynyl, alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl,cycloalkyl alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl,peptidyl, polyamino, or polyalkyleneoxy. In some embodiments, the cyclicbis-electrophile can be defined by Formula IIA, Formula IIB, or FormulaIIC below

wherein LG and Z are as defined above with respect to Formula II. Thepolynucleophilic monomer can be defined by Formula III below

wherein A represents a heterocyclic ring comprising a tertiary nitrogenatom; Y is absent or represents a linking group; and R¹ is absent, orrepresents halogen, hydroxy, amino, cyano, azido, hydrazone, alkyl,alkenyl, alkynyl, alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl,cycloalkyl alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, dialkylamino,alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,peptidyl, polyamino, or polyalkyleneoxy.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the ¹H NMR spectra of WCL electrophiles. The inset showsthe C_(1/5)-H ¹H NMR resonances of equimolar mixtures of pairs ofelectrophiles after reaction with 0.5 equiv. of benzylamine, confirmingthe reactivity order iodide>nitrate>chloride.

FIG. 2 shows the ¹H NMR analysis of competition experiments between WCLnitrate and chloride electrophiles for benzylamine.

FIG. 3 shows the ¹H NMR analysis of competition experiments between WCLchloride, nitrate, and iodide electrophiles for benzylamine.

FIGS. 4A-4B show ¹H NMR spectra taken at various intervals over 100hours as 5 d was incubated in deuterated aqueous phosphate buffer (pH 7)at 50° C. FIG. 4A shows the downfield region of the ¹H NMR spectra. FIG.4B shows the upfield region of the ¹H NMR spectra.

FIG. 5 shows the pseudo-first order plot obtained for the fragmentationof 5 d in deuterated buffer at 50° C.

FIG. 6 shows ¹H NMR spectra taken at various intervals over 204 hours aspolymer 3i was incubated in deuterium oxide at 37° C.

FIG. 7A shows gel permeation chromatography (GPC) traces of polymer 3abefore (black trace) and after (gray trace) fragmentation in water at37° C.

FIG. 7B shows GPC traces of polymer 3c before (black trace) and after(gray trace) fragmentation in water at 37° C.

FIG. 7C shows GPC traces of polymer 3i before (black trace) and after(gray trace) fragmentation in water at 37° C.

FIG. 8A shows the results of thermogravimetric analysis (TGA) ofpolymers 3a, 3c, and 3i.

FIG. 8B shows a differential scanning calorimetry (DSC) trace obtainedfor polymer 3a.

FIG. 9A shows a box-and-whisker plot of viability test results forGFP-HeLa at different N/P ratios with polymer 3a.

FIG. 9B shows a box-and-whisker plot of viability test results forGFP-HeLa at different N/P ratios with polymer 3i.

FIG. 10 shows a box-and-whisker plot summarizing the results of siRNAtransfection tests at various N/P ratios with polymer 3a.

FIG. 11A is a plot of cytotoxicity tests of polymer 3a towards CHO-K1.Cell viability was measured after 4 h (black bars) or 24 h (red bars)treatment at the indicated concentration.

FIG. 11B is a plot of cytotoxicity tests of polymer 3c towards CHO-K1.Cell viability was measured after 4 h (black bars) or 24 h (red bars)treatment at the indicated concentration.

FIG. 11C is a plot of cytotoxicity tests of polymer 3i towards CHO-K1.Cell viability was measured after 4 h (black bars) or 24 h (red bars)treatment at the indicated concentration.

FIG. 11D is a plot of cytotoxicity tests of polymer 3a′ towards CHO-K1.Cell viability was measured after 4 h (black bars) or 24 h (red bars)treatment at the indicated concentration.

FIG. 12A is a plot of cytotoxicity tests of monomer 2a towards CHO-K1.Cell viability was measured after 4 h (black bars) or 24 h (red bars)treatment at the indicated concentration.

FIG. 12B is a plot of cytotoxicity tests of monomer 2c towards CHO-K1.Cell viability was measured after 4 h (black bars) or 24 h (red bars)treatment at the indicated concentration.

FIG. 12C is a plot of cytotoxicity tests of monomer 2i towards CHO-K1.Cell viability was measured after 4 h (black bars) or 24 h (red bars)treatment at the indicated concentration.

FIG. 12D is a plot of cytotoxicity tests of monomer 4 towards CHO-K1.Cell viability was measured after 4 h (black bars) or 24 h (red bars)treatment at the indicated concentration.

FIG. 12E is a plot of cytotoxicity tests of compound 5a towards CHO-K1.Cell viability was measured after 4 h (black bars) or 24 h (red bars)treatment at the indicated concentration.

FIG. 12F is a plot of cytotoxicity tests of compound 5c towards CHO-K1.Cell viability was measured after 4 h (black bars) or 24 h (red bars)treatment at the indicated concentration.

FIG. 12G is a plot of cytotoxicity tests of compound 5i towards CHO-K1.Cell viability was measured after 4 h (black bars) or 24 h (red bars)treatment at the indicated concentration.

FIG. 12H is a plot of cytotoxicity tests of compound 5m towards CHO-K1.Cell viability was measured after 4 h (black bars) or 24 h (red bars)treatment at the indicated concentration.

FIG. 13A is a plot of the adduct half life at 50° C. vs. pyridinebasicity (see also Table 1). The pyridine basicity is indicated by thecalculated pK_(a) of the conjugate acid. The dotted line is meant tohighlight the approximate trend, and is not a mathematical fit.

FIG. 13B is a plot of the adduct half life at 37° C. vs. pyridinebasicity (see also Table 2). The pyridine basicity is indicated by thecalculated pK_(a) of the conjugate acid. The dotted line is meant tohighlight the approximate trend, and is not a mathematical fit.

FIG. 14 shows the ¹H NMR spectrum of an oligomer formed by reaction of1b and 2a (Table 4, entry 3).

FIG. 15A shows the results of agarose gel electrophoresis of plasmid DNAmixed with increasing amounts of polycation 3a.

FIG. 15B shows the results of dynamic light scattering of a PBS buffersolution containing plasmid DNA mixed with polycation 3a at an N/P ratioof 2/1.

FIG. 16 is an assessment of the cytotoxic activity of examplepolycations (MIC=minimum concentration required to inhibit the growth of90% of cells in a standard starting culture). Cytotoxicity was evaluatedagainst B. subtilis (a representative Gram-positive strain), E. Coli (arepresentative Gram-negative strain), and CHO-K1 (a representativemammalian cell). Certain commercially-available antibacterial agents(e.g., amoxicillin) are shown for reference. Initial studies suggestthat the polycations can exhibit broadspectrum activity against bacteriawith a large therapeutic window.

FIG. 17 is a schematic illustration of the methods used to assess thecytotoxicity of glass substrates covalently functionalized with examplepolycations.

FIG. 18 is a schematic illustration of methods for covalentlyfunctionalizing polyvinyl chloride (e.g., medical tubing) to improveresistance to bacterial and fungal colonization and biofilm formation.

FIG. 19 is a schematic illustrated of classes of polycationic materialsbased on the reaction of a 9-thia/aza/selenabicyclo[3.3.1]nonylelectrophile with a nucleophile.

FIG. 20 is a schematic illustration of the strategy used to immobilizethe polycations on a glass substrate.

FIG. 21 is a schematic illustration of the strategy used to immobilizethe polycations on a PVC substrate.

DETAILED DESCRIPTION

The materials, compounds, compositions, articles, and methods describedherein may be understood more readily by reference to the followingdetailed description of specific aspects of the disclosed subject matterand the Examples and Figures included therein.

Before the present materials, compounds, compositions, and methods aredisclosed and described, it is to be understood that the aspectsdescribed below are not limited to specific synthetic methods orspecific reagents, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to be limiting.

Also, throughout this specification, various publications arereferenced. The disclosures of these publications in their entiretiesare hereby incorporated by reference into this application in order tomore fully describe the state of the art to which the disclosed matterpertains. The references disclosed are also individually andspecifically incorporated by reference herein for the material containedin them that is discussed in the sentence in which the reference isrelied upon.

General Definitions

In this specification and in the claims that follow, reference will bemade to a number of terms, which shall be defined to have the followingmeanings:

Throughout the description and claims of this specification the word“comprise” and other forms of the word, such as “comprising” and“comprises,” means including but not limited to, and is not intended toexclude, for example, other additives, components, integers, or steps.

As used in the description and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a composition”includes mixtures of two or more such compositions, reference to “thecompound” includes mixtures of two or more such compounds, reference to“an agent” includes mixture of two or more such agents, and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

By “reduce” or other forms of the word, such as “reducing” or“reduction,” is meant lowering of an event or characteristic (e.g.,bacterial growth, biofilm formation, etc.). It is understood that thisis typically in relation to some standard or expected value, in otherwords it is relative, but that it is not always necessary for thestandard or relative value to be referred to. For example, “reducesbiofilm formation” means reducing the rate of biofilm formation relativeto a standard or a control.

By “prevent” or other forms of the word, such as “preventing” or“prevention,” is meant to stop a particular event or characteristic, tostabilize or delay the development or progression of a particular eventor characteristic, or to minimize the chances that a particular event orcharacteristic will occur. Prevent does not require comparison to acontrol as it is typically more absolute than, for example, reduce. Asused herein, something could be reduced but not prevented, but somethingthat is reduced could also be prevented.

Likewise, something could be prevented but not reduced, but somethingthat is prevented could also be reduced. It is understood that wherereduce or prevent are used, unless specifically indicated otherwise, theuse of the other word is also expressly disclosed.

“Contacting,” as used herein, refers to any means for providing acomponent or element to a surface. Contacting can include, for example,spraying, wetting, immersing, dipping, painting, flowing across, bondingor adhering or otherwise providing a surface with a the component orelement.

“Biofilm” or “biofilms” refer to communities of microorganisms that areattached to a substrate. The microorganisms often excrete a protectiveand adhesive matrix of polymeric compounds. They often have structuralheterogeneity, genetic diversity, and complex community interactions.“Biofilm preventing”, “biofilm removing”, “biofilm inhibiting”, “biofilmreducing”, “biofilm resistant”, “biofilm controlling” or “antifouling”refer to prevention of biofilm formation, inhibition of theestablishment or growth of a biofilm, or decrease in the amount oforganisms that attach and/or grow upon a substrate, up to and includingthe complete removal of the biofilm.

The phrase “effective amount” or “amount effective” refers to an amountof an antimicrobial agent (e.g., a polycationic polymer describedherein) that significantly reduces the number of organisms that attachto a defined surface (cells/mm²) relative to the number that attach toan untreated surface. Particularly preferred are amounts that reduce thenumber of organisms that attach to the surface by a factor of at least2. Even more preferred are amounts that reduce the surface attachment oforganisms by a factor of 4, more preferably by a factor of 6. Aneffective amount of an antimicrobial agent (e.g., a polycationic polymerdescribed herein) is said to inhibit the formation of biofilms, and toinhibit the growth of organisms on a defined surface. The term“inhibit,” as applied to the effect of an antimicrobial agent (e.g., apolycationic polymer described herein) on a surface includes any actionthat significantly reduces the number of organisms that attach thereto.

It is understood that throughout this specification the identifiers“first” and “second” are used solely to aid in distinguishing thevarious components and steps of the disclosed subject matter. Theidentifiers “first” and “second” are not intended to imply anyparticular order, amount, preference, or importance to the components orsteps modified by these terms.

Chemical Definitions

Terms used herein will have their customary meaning in the art unlessspecified otherwise. The organic moieties mentioned when definingvariable positions within the general formulae described herein (e.g.,the term “halogen”) are collective terms for the individual substituentsencompassed by the organic moiety. The prefix C_(n)-C_(m) indicates ineach case the possible number of carbon atoms in the group.

The term “alkyl,” as used herein, refers to saturated straight,branched, primary, secondary or tertiary hydrocarbons, including thosehaving 1 to 20 atoms. In some examples, alkyl groups will includeC₁-C₁₂, C₁-C₁₀, C₁-C₈, C₁-C₆, C₁-C₅, C₁-C₄, C₁-C₃, or C₁-C₂ alkylgroups. Examples of C₁-C₁₀ alkyl groups include, but are not limited to,methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl,2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl,3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl,1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl,3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl,1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl,3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl,1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl,heptyl, octyl, 2-ethylhexyl, nonyl and decyl groups, as well as theirisomers. Examples of C₁-C₄-alkyl groups include, for example, methyl,ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, and1,1-dimethylethyl groups.

Optionally, alkyl groups may also contain one or more heteroatoms withinthe carbon backbone. The heteroatoms incorporated into the carbonbackbone may be oxygen, nitrogen, sulfur, or combinations thereof. Incertain embodiments, the alkyl group can include between one and fourheteroatoms.

Cyclic alkyl groups or “cycloalkyl” groups include cycloalkyl groupshaving from 3 to 10 carbon atoms. Cycloalkyl groups can include a singlering, or multiple condensed rings. In some examples, cycloalkyl groupsinclude C₃-C₄, C₄-C₇, C₅-C₇, C₄-C₆, or C₅-C₆ cyclic alkyl groups.Non-limiting examples of cycloalkyl groups include adamantyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl and the like.

Alkyl and cycloalkyl groups can be unsubstituted or substituted with oneor more moieties chosen from alkyl, halo, haloalkyl, hydroxyl, carboxyl,acyl, acyloxy, amino, alkyl- or dialkylamino, amido, arylamino, alkoxy,aryloxy, nitro, cyano, azido, thiol, imino, sulfonic acid, sulfate,sulfonyl, sulfanyl, sulfinyl, sulfamonyl, ester, phosphonyl, phosphinyl,phosphoryl, phosphine, thioester, thioether, acid halide, anhydride,oxime, hydrazine, carbamate, phosphoric acid, phosphate, phosphonate,hydrazone, carbonate, ammonium (e.g., a tetraalkylammonium group, aaryltrialkylammonium group, a diaryldialkylammonium group, or atriarylalkylammonium group), pyridinium, or any other viable functionalgroup that does not inhibit the biological activity of the compounds ofthe invention, either unprotected, or protected as necessary, as knownto those skilled in the art, for example, as described in Greene, etal., Protective Groups in Organic Synthesis, John Wiley and Sons, ThirdEdition, 1999, hereby incorporated by reference.

Terms including the term “alkyl,” such as “alkylamino” or“dialkylamino,” will be understood to comprise an alkyl group as definedabove linked to another functional group, where the group is linked tothe compound through the last group listed, as understood by those ofskill in the art.

The term “alkenyl,” as used herein, refers to both straight and branchedcarbon chains which have at least one carbon-carbon double bond. In someexamples, alkenyl groups can include C₂-C₂₀ alkenyl groups. In otherexamples, alkenyl can include C₂-C₁₂, C₂-C₁₀, C₂-C₈, C₂-C₆, or C₂-C₄alkenyl groups. In one embodiment of alkenyl, the number of double bondsis 1-3, in another embodiment of alkenyl, the number of double bonds isone or two. Other ranges of carbon-carbon double bonds and carbonnumbers are also contemplated depending on the location of the alkenylmoiety on the molecule. “C₂-C₁₀-alkenyl” groups can include more thanone double bond in the chain. The one or more unsaturations within thealkenyl group can be located at any position(s) within the carbon chainas valence permits. In some examples, when the alkenyl group iscovalently bound to one or more additional moieties, the carbon atom(s)in the alkenyl group that are covalently bound to the one or moreadditional moieties are not part of a carbon-carbon double bond withinthe alkenyl group. Examples of alkenyl groups include, but are notlimited to, ethenyl, 1-propenyl, 2-propenyl, 1-methyl-ethenyl,1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-1-propenyl,2-methyl-1-propenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl;1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl,2-methyl-1-butenyl, 3-methyl-1-butenyl, 1-methyl-2-butenyl,2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl,2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl,1,2-dimethyl-1-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-1-propenyl,1-ethyl-2-propenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl,5-hexenyl, 1-methyl-1-pentenyl, 2-methyl-1-pentenyl,3-methyl-1-pentenyl, 4-methyl-1-pentenyl, 1-methyl-2-pentenyl,2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl,1-methyl-3-pentenyl, 2-methyl-3-pentenyl, 3-methyl-3-pentenyl,4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl,3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl,1,1-dimethyl-3-butenyl, 1,2-dimethyl-1-butenyl, 1,2-dimethyl-2-butenyl,1,2-dimethyl-3-butenyl, 1,3-dimethyl-1-butenyl, 1,3-dimethyl-2-butenyl,1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, 2,3-dimethyl-1-butenyl,2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 3,3-dimethyl-1-butenyl,3,3-dimethyl-2-butenyl, 1-ethyl-1-butenyl, 1-ethyl-2-butenyl,1-ethyl-3-butenyl, 2-ethyl-1-butenyl, 2-ethyl-2-butenyl,2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl,1-ethyl-1-methyl-2-propenyl, 1-ethyl-2-methyl-1-propenyl and1-ethyl-2-methyl-2-propenyl groups.

The term “alkynyl,” as used herein, refers to both straight and branchedcarbon chains which have at least one carbon-carbon triple bond. In oneembodiment of alkynyl, the number of triple bonds is 1-3; in anotherembodiment of alkynyl, the number of triple bonds is one or two. In someexamples, alkynyl groups include from C₂-C₂₀ alkynyl groups. In otherexamples, alkynyl groups can include C₂-C₁₂, C₂-C₁₀, C₂-C₈, C₂-C₆ orC₂-C₄ alkynyl groups. Other ranges of carbon-carbon triple bonds andcarbon numbers are also contemplated depending on the location of thealkenyl moiety on the molecule. For example, the term “C₂-C₁₀-alkynyl”as used herein refers to a straight-chain or branched unsaturatedhydrocarbon group having 2 to 10 carbon atoms and containing at leastone triple bond, such as ethynyl, prop-1-yn-1-yl, prop-2-yn-1-yl,n-but-1-yn-1-yl, n-but-1-yn-3-yl, n-but-1-yn-4-yl, n-but-2-yn-1-yl,n-pent-1-yn-1-yl, n-pent-1-yn-3-yl, n-pent-1-yn-4-yl, n-pent-1-yn-5-yl,n-pent-2-yn-1-yl, n-pent-2-yn-4-yl, n-pent-2-yn-5-yl,3-methylbut-1-yn-3-yl, 3-methylbut-1-yn-4-yl, n-hex-1-yn-1-yl,n-hex-1-yn-3-yl, n-hex-1-yn-4-yl, n-hex-1-yn-5-yl, n-hex-1-yn-6-yl,n-hex-2-yn-1-yl, n-hex-2-yn-4-yl, n-hex-2-yn-5-yl, n-hex-2-yn-6-yl,n-hex-3-yn-1-yl, n-hex-3-yn-2-yl, 3-methylpent-1-yn-1-yl,3-methylpent-1-yn-3-yl, 3-methylpent-1-yn-4-yl, 3-methylpent-1-yn-5-yl,4-methylpent-1-yn-1-yl, 4-methylpent-2-yn-4-yl, and4-methylpent-2-yn-5-yl groups.

Alkenyl and alkynyl groups can be unsubstituted or substituted with oneor more moieties chosen from alkyl, halo, haloalkyl, hydroxyl, carboxyl,acyl, acyloxy, amino, alkyl- or dialkylamino, amido, arylamino, alkoxy,aryloxy, nitro, cyano, azido, thiol, imino, sulfonic acid, sulfate,sulfonyl, sulfanyl, sulfinyl, sulfamonyl, ester, phosphonyl, phosphinyl,phosphoryl, phosphine, thioester, thioether, acid halide, anhydride,oxime, hydrazine, carbamate, phosphoric acid, phosphate, phosphonate,hydrazone, carbonate, ammonium (e.g., a tetraalkylammonium group, aaryltrialkylammonium group, a diaryldialkylammonium group, or atriarylalkylammonium group), pyridinium, or any other viable functionalgroup that does not inhibit the biological activity of the compounds ofthe invention, either unprotected, or protected as necessary, as knownto those skilled in the art, for example, as described in Greene, etal., Protective Groups in Organic Synthesis, John Wiley and Sons, ThirdEdition, 1999.

The term “aryl,” as used herein, refers to a monovalent aromaticcarbocyclic group of from 6 to 14 carbon atoms. Aryl groups can includea single ring or multiple condensed rings. In some examples, aryl groupsinclude C₆-C₁₀ aryl groups. Aryl groups include, but are not limited to,phenyl, biphenyl, naphthyl, tetrahydronaphtyl, phenylcyclopropyl andindanyl. Aryl groups can be unsubstituted or substituted by one or moremoieties chosen from halo, cyano, nitro, hydroxy, mercapto, amino,alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, haloalkyl,haloalkenyl, haloalkynyl, halocycloalkyl, halocycloalkenyl, alkoxy,alkenyloxy, alkynyloxy, haloalkoxy, haloalkenyloxy, haloalkynyloxy,cycloalkoxy, cycloalkenyloxy, halocycloalkoxy, halocycloalkenyloxy,alkylthio, haloalkylthio, cycloalkylthio, halocycloalkylthio,alkylsulfinyl, alkenylsulfinyl, alkynyl-sulfinyl, haloalkylsulfinyl,haloalkenylsulfinyl, haloalkynylsulfinyl, alkylsulfonyl,alkenylsulfonyl, alkynylsulfonyl, haloalkyl-sulfonyl,haloalkenylsulfonyl, haloalkynylsulfonyl, alkylamino, alkenylamino,alkynylamino, di(alkyl)amino, di(alkenyl)-amino, di(alkynyl)amino, ortrialkylsilyl.

The term “alkylaryl,” as used herein, refers to an aryl group that isbonded to a parent compound through a diradical alkylene bridge,(—CH₂—)_(n), where n is 1-12 (e.g., n is from 1 to 6) and where “aryl”is as defined above. The term “arylalkyl,” as used herein, refers to anaryl group, as defined above, which is substituted by an alkyl group, asdefined above.

The term “alkylcycloalkyl,” as used herein, refers to a cycloalkyl groupthat is bonded to a parent compound through a diradical alkylene bridge,(—CH₂—)_(n), where n is 1-12 (e.g., n is from 1 to 6) and where“cycloalkyl” is as defined above.

The term “alkoxy,” as used herein, refers to alkyl-O—, wherein alkylrefers to an alkyl group, as defined above. Similarly, the terms“alkenyloxy,” “alkynyloxy,” and “cycloalkoxy,” refer to the groupsalkenyl-O—, alkynyl-O—, and cycloalkyl-O—, respectively, whereinalkenyl, alkynyl, and cycloalkyl are as defined above. Examples ofC₁-C₆-alkoxy groups include, but are not limited to, methoxy, ethoxy,C₂H₅—CH₂O—, (CH₃)₂CHO—, n-butoxy, C₂H₅—CH(CH₃)O—, (CH₃)₂CH—CH₂O—,(CH₃)₃CO—, n-pentoxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy,1,1-dimethylpropoxy, 1,2-dimethylpropoxy, 2,2-dimethyl-propoxy,1-ethylpropoxy, n-hexoxy, 1-methylpentoxy, 2-methylpentoxy,3-methylpentoxy, 4-methylpentoxy, 1,1-dimethylbutoxy,1,2-dimethylbutoxy, 1,3-dimethylbutoxy, 2,2-dimethylbutoxy,2,3-dimethylbutoxy, 3,3-dimethylbutoxy, 1-ethylbutoxy, 2-ethylbutoxy,1,1,2-trimethylpropoxy, 1,2,2-trimethylpropoxy, 1-ethyl-1-methylpropoxy,and 1-ethyl-2-methylpropoxy.

The term “alkylthio,” as used herein, refers to alkyl-S—, wherein alkylrefers to an alkyl group, as defined above. Similarly, the term“cycloalkylthio,” refers to cycloalkyl-S— where cycloalkyl are asdefined above.

The term “alkylsulfinyl,” as used herein, refers to alkyl-S(O)—, whereinalkyl refers to an alkyl group, as defined above.

The term “alkylsulfonyl,” as used herein, refers to alkyl-S(O)₂—,wherein alkyl is as defined above.

The terms “alkylamino” and “dialkylamino,” as used herein, refer toalkyl-NH— and (alkyl)₂N— groups, where alkyl is as defined above.

The terms “alkylcarbonyl,” “alkoxycarbonyl,” “alkylaminocarbonyl,” and“dialkylaminocarbonyl,” as used herein, refer to alkyl-C(O)—,alkoxy-C(O)—, alkylamino-C(O)— and dialkylamino-C(O)— respectively,where alkyl, alkoxy, alkylamino, and dialkylamino are as defined above.

The term “heteroaryl,” as used herein, refers to a monovalent aromaticgroup of from 1 to 15 carbon atoms (e.g., from 1 to 10 carbon atoms,from 2 to 8 carbon atoms, from 3 to 6 carbon atoms, or from 4 to 6carbon atoms) having one or more heteroatoms within the ring. Theheteroaryl group can include from 1 to 4 heteroatoms, from 1 to 3heteroatoms, or from 1 to 2 heteroatoms. In some examples, theheteroatom(s) incorporated into the ring are oxygen, nitrogen, sulfur,or combinations thereof. When present, the nitrogen and sulfurheteroatoms can optionally be oxidized. Heteroaryl groups can have asingle ring (e.g., pyridyl or furyl) or multiple condensed ringsprovided that the point of attachment is through a heteroaryl ring atom.Preferred heteroaryls include pyridyl, piridazinyl, pyrimidinyl,pyrazinyl, triazinyl, pyrrolyl, indolyl, quinolinyl, isoquinolinyl,quinazolinyl, quinoxalinnyl, furanyl, thiophenyl, furyl, pyrrolyl,imidazolyl, oxazolyl, isoxazolyl, isothiazolyl, pyrazolyl benzofuranyl,and benzothiophenyl. Heteroaryl rings can be unsubstituted orsubstituted by one or more moieties as described for aryl above.

The term “alkylheteroaryl,” as used herein, refers to a heteroaryl groupthat is bonded to a parent compound through a diradical alkylene bridge,(—CH₂—)_(n), where n is 1-12 and where “heteroaryl” is as defined above.

The terms “heterocyclyl,” “heterocyclic” and “heterocyclo” are usedherein interchangeably, and refer to fully saturated or unsaturated,cyclic groups, for example, 3 to 7 membered monocyclic or 4 to 7membered monocyclic; 7 to 11 membered bicyclic, or 10 to 15 memberedtricyclic ring systems, having one or more heteroatoms within the ring.The heterocyclyl group can include from 1 to 4 heteroatoms, from 1 to 3heteroatoms, or from 1 to 2 heteroatoms. In some examples, theheteroatom(s) incorporated into the ring are oxygen, nitrogen, sulfur,or combinations thereof. When present, the nitrogen and sulfurheteroatoms can optionally be oxidized, and the nitrogen heteroatoms canoptionally be quaternized. The heterocyclyl group can be attached at anyheteroatom or carbon atom of the ring or ring system and can beunsubstituted or substituted by one or more moieties as described foraryl groups above.

Exemplary monocyclic heterocyclic groups include, but are not limitedto, pyrrolidinyl, pyrrolyl, pyrazolyl, oxetanyl, pyrazolinyl,imidazolyl, imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl,isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl,isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, thienyl,oxadiazolyl, piperidinyl, piperazinyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl,4-piperidonyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl,tetrahydropyranyl, morpholinyl, thiamorpholinyl, thiamorpholinylsulfoxide, thiamorpholinyl sulfone, 1,3-dioxolane andtetrahydro-1,1-dioxothienyl, triazolyl, triazinyl, and the like.

Exemplary bicyclic heterocyclic groups include, but are not limited to,indolyl, benzothiazolyl, benzoxazolyl, benzodioxolyl, benzothienyl,quinuclidinyl, quinolinyl, tetra-hydroisoquinolinyl, isoquinolinyl,benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, chromonyl,coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl,pyrrolopyridyl, furopyridinyl (such as furo[2,3-c]pyridinyl,furo[3,2-b]pyridinyl]or furo[2,3-b]pyridinyl), dihydroisoindolyl,dihydroquinazolinyl (such as 3,4-dihydro-4-oxo-quinazolinyl),tetrahydroquinolinyl and the like.

Exemplary tricyclic heterocyclic groups include carbazolyl, benzidolyl,phenanthrolinyl, acridinyl, phenanthridinyl, xanthenyl, and the like.

The term “alkylheterocyclyl,” as used herein, refers to a heterocyclylgroup that is bonded to a parent compound through a diradical alkylenebridge, (—CH₂—)_(n), where n is 1-12 and where “heterocyclyl” is asdefined above. The term “heterocyclylalkyl,” as used herein, refers to aheterocyclyl group, as defined above, which is substituted by an alkylgroup, as defined above.

Heretrocyclyl and heteroaryl groups can be unsubstituted or substitutedwith one or more moieties chosen from alkyl, halo, haloalkyl, hydroxyl,carboxyl, acyl, acyloxy, amino, alkyl- or dialkylamino, amido,arylamino, alkoxy, aryloxy, nitro, cyano, azido, thiol, imino, sulfonicacid, sulfate, sulfonyl, sulfanyl, sulfinyl, sulfamonyl, ester,phosphonyl, phosphinyl, phosphoryl, phosphine, thioester, thioether,acid halide, anhydride, oxime, hydrazine, carbamate, phosphoric acid,phosphate, phosphonate, hydrazone, carbonate, ammonium (e.g., atetraalkylammonium group, a aryltrialkylammonium group, adiaryldialkylammonium group, or a triarylalkylammonium group),pyridinium, or any other viable functional group that does not inhibitthe biological activity of the compounds of the invention, eitherunprotected, or protected as necessary, as known to those skilled in theart, for example, as described in Greene, et al., Protective Groups inOrganic Synthesis, John Wiley and Sons, Third Edition, 1999

The term “halogen,” as used herein, refers to the atoms fluorine,chlorine, bromine and iodine. The prefix halo- (e.g., as illustrated bythe term haloalkyl) refers to all degrees of halogen substitution, froma single substitution to a perhalo substitution (e.g., as illustratedwith methyl as chloromethyl (—CH₂Cl), dichloromethyl (—CHCl₂),trichloromethyl (—CCl₃)).

The term “polyalkyleneoxy,” as used herein, generally refers to anoligomeric or polymeric group formed from the following repeatingalkyleneoxy units: —CH₂CH₂O—, CH₂CH₂CH₂O—, —CH₂CH₂CH₂CH₂O—,—CH₂CH(CH₃)O—, —CH₂CH(CH₂CH₃)O—CH₂CH₂CH(CH₃)O—, and any combinationthereof. The number of repeat alkyleneoxy groups typically is from 2 to50 (e.g., from 5 to 50, from 10 to 50, from 2 to 40, from 5 to 40, from10 to 40, from 2 to 30, from 5 to 30, from 10 to 30, from 2 to 20, from5 to 20, from 10 to 20, from 2 to 15, from 5 to 15, from 10 to 15, from2 to 10, or from 5 to 10) repeat alkyleneoxy groups, although the numberof repeat units can be outside of these ranges. The term “pendant”, asused herein in reference to a polyalkyleneoxy group, refers to apolyalkyleneoxy group that is present as an end group and/or a sidechainattached to a polymeric backbone.

The term “polyamino,” as used herein, generally refers to an oligomericor polymeric group derived from one or more monomers containing an aminegroup. Suitable monomers of this type include vinylamine, allylamine,and ethyleneimine Other suitable amino-containing monomers include(meth)acrylate monomers containing one or more primary and/or secondaryamine groups, such as 2-aminoethyl methacrylate, 2-aminoethyl acrylate,2-(tert-butylamino)ethyl acrylate, 2-(tert-butylamino)ethylmethacrylate. In some cases, the polyamino group can be a polycationicpolyamino group (e.g., polyethyleneimine) The term “pendant”, as usedherein in reference to a polyamino group, refers to a polyamino groupthat is present as an end group and/or a sidechain attached to apolymeric backbone.

As used herein, the term “(meth)acrylate monomer” includes acrylate,methacrylate, diacrylate, and dimethacrylate monomers.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, and aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, Such substituents include, but are not limited to, halogen,hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or anacyl), thiocarbonyl (such as a thioester, a thioacetate, or athioformate), alkoxyl, phosphoryl, phosphate, phosphonate, aphosphinate, amino, amido, amidine, imine, cyano, nitro, azido,sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido,sulfonyl, heterocyclyl, aralkyl, hydrazone, carbonate, ammonium (e.g., atetraalkylammonium group, a aryltrialkylammonium group, adiaryldialkylammonium group, or a triarylalkylammonium group),pyridinium, or an aromatic or heteroaromatic moiety.

The permissible substituents can be one or more and the same ordifferent for appropriate organic compounds. For purposes of thisdisclosure, the heteroatoms, such as nitrogen, can have hydrogensubstituents and/or any permissible substituents of organic compoundsdescribed herein which satisfy the valences of the heteroatoms. Thisdisclosure is not intended to be limited in any manner by thepermissible substituents of organic compounds. Also, the terms“substitution” or “substituted with” include the implicit proviso thatsuch substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., a compound that does not spontaneouslyundergo transformation such as by rearrangement, cyclization,elimination, etc.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g., each enantiomer, diastereomer, and meso compound,and a mixture of isomers, such as a racemic or scalemic mixture.

Reference will now be made in detail to specific aspects of thedisclosed materials, compounds, compositions, articles, and methods,examples of which are illustrated in the accompanying Examples andFigures.

Processes

Provided herein are processes for rendering a polyvinyl chloride (PVC)surface antimicrobial (e.g., antibacterial and/or antifungal). Theprocesses generally involve functionalization of the PVC surface toconvert chloride residues on the PVC surface to functional groups (e.g.,an alkyne or an azide) that can participate in a click reaction (e.g., a1,3-dipolar cycloaddition reaction). Subsequently, click chemistry canbe used to covalently tether one or more antimicrobial agents to thesurface. The resulting surfaces can exhibit antimicrobial activity(e.g., antibacterial activity, antifungal activity, or a combinationthereof).

Importantly, these processes can employ mild reaction conditions thatpermit covalent attachment of the antimicrobial agents to a PVC surfacewithout substantially altering the physiochemical and mechanicalproperties of the article comprising the PVC surface. As a consequence,the processes described herein can be used to impart antimicrobialactivity to a surface of a formed PVC article (e.g., an extruded articlesuch as PVC tubing) without unacceptably compromising the physiochemicaland mechanical properties of the article (e.g., without damaging the PVCtubing).

In some embodiments, the process for rendering a polyvinyl chloridesurface antimicrobial can comprise (a) contacting the polyvinyl chloridewith an azidation reagent in the presence of a phase transfer catalystto form an azide-substituted polyvinyl chloride; and (b) contacting theazide-substituted polyvinyl chloride with an antimicrobial agentcomprising an alkyne moiety under conditions effective to covalentlybond the antimicrobial agent to the polyvinyl chloride surface.

In some embodiments, step (a) can comprise flowing an aqueous solutioncomprising the azidation reagent and the phase transfer catalyst acrossthe polyvinyl chloride surface. This can be accomplished, for example,using a flow reactor. By way of example, in the case of a segment of PVCtubing, step (a) can comprise flowing an aqueous solution comprising theazidation reagent and the phase transfer catalyst through the segment ofPVC tubing to form an azide-substituted polyvinyl chloride on theinterior surface of the PVC tubing. Step (a) can also involve, forexample, immersing the PVC surface in an aqueous solution comprising theazidation reagent and the phase transfer catalyst, or spraying anaqueous solution comprising the azidation reagent and the phase transfercatalyst onto the PVC surface.

In some embodiments, step (a) can be performed at a temperature of from20° C. to 25° C. In some embodiments, the reaction time in step (a) canbe limited in order to minimize any potential impact on thephysiochemical and/or mechanical properties of the article comprisingthe PVC surface. For example, in some embodiments, step (a) can comprisecontacting the polyvinyl chloride with the azidation reagent and thephase transfer catalyst for 8 hours or less (e.g., 7 hours or less, 6hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2hours or less, or 1 hour or less). In certain embodiments, step (a) cancomprise contacting the polyvinyl chloride with the azidation reagentand the phase transfer catalyst for from 10 minutes to 8 hours (e.g.,from 10 minutes to 6 hours, from 10 minutes to 3 hours, from 10 minutesto 2 hours, or from 10 minutes to 1 hour).

The phase transfer catalyst can comprise a salt compound, such as aGroup 15 salt compound (e.g., a quaternary ammonium salt, or aphosphonium salt). Examples of suitable phase transfer catalysts include(but are not limited to) tetrabutyl ammonium bromide, tetrabutylammonium cyanide, tetrabutyl ammonium iodide, tetrabutyl ammoniumnitrate, tetrabutyl ammonium bisulfate, tetrabutyl phosphonium bromide,tetraphenyl phosphonium bromide, or a combination thereof. In someembodiments, the phase transfer catalyst can comprise one of thefollowing:

Examples of suitable azidation reagents include, for example, sodiumazide (NaN₃), trimethylsilyl azide, diphenylphosphoryl azide, potassiumazide, chlorine azide, bromine azide, iodine azide, hydrazoic acid,tetraalkylammonium azides, tetraphenylphosphonium azides, andcombinations thereof. In certain embodiments, the azidation reagent cancomprise NaN₃.

In some embodiments, step (b) can be performed at a temperature of from20° C. to 25° C. In some embodiments, step (a) can comprise flowing anaqueous solution comprising the antimicrobial agent comprising thealkyne moiety across the azide-substituted polyvinyl chloride surface.As described above, this can be accomplished, for example, using a flowreactor. By way of example, in the case of a segment of PVC tubing, step(b) can comprise flowing an aqueous solution comprising theantimicrobial agent comprising the alkyne moiety through the segment ofPVC tubing that includes the azide-substituted polyvinyl chloridesurface. Step (b) can also involve, for example, immersing theazide-substituted PVC surface in an aqueous solution comprising theantimicrobial agent, or spraying an aqueous solution comprising theantimicrobial agent onto the azide-substituted PVC surface.

In some embodiments, the alkyne moiety present on the antimicrobialagent can be activated (e.g., activated by ring strain, activated by oneor more electron withdrawing groups, or a combination thereof). In someof these embodiments, the step (b) can be performed without a catalyst.Alternatively, a Cu(I) catalyst may be used to facilitate reactionbetween the azide on the PVC surface and the alkyne moiety on theantimicrobial agent. In these embodiments, step (b) can comprisecontacting azide-substituted polyvinyl chloride with the antimicrobialagent comprising the alkyne moiety in the presence of a Cu(I) catalyst.

In other embodiments, the process for rendering a polyvinyl chloridesurface antimicrobial can comprise (a) contacting the polyvinyl chloridewith a cyanation reagent in the presence of a phase transfer catalyst toform a cyano-substituted polyvinyl chloride; and (b) covalently bondingan antimicrobial agent to the cyano-substituted polyvinyl chloride.

In some embodiments, step (a) can comprise flowing an aqueous solutioncomprising the cyanation reagent and the phase transfer catalyst acrossthe polyvinyl chloride surface. This can be accomplished, for example,using a flow reactor. By way of example, in the case of a segment of PVCtubing, step (a) can comprise flowing an aqueous solution comprising thecyanation reagent and the phase transfer catalyst through the segment ofPVC tubing to form an cyano-substituted polyvinyl chloride on theinterior surface of the PVC tubing. Step (a) can also involve, forexample, immersing the PVC surface in an aqueous solution comprising thecyanation reagent and the phase transfer catalyst, or spraying anaqueous solution comprising the cyanation reagent and the phase transfercatalyst onto the PVC surface.

In some embodiments, step (a) can be performed at a temperature of from20° C. to 25° C. In some embodiments, the reaction time in step (a) canbe limited in order to minimize any potential impact on thephysiochemical and/or mechanical properties of the article comprisingthe PVC surface. For example, in some embodiments, step (a) can comprisecontacting the polyvinyl chloride with the cyanation reagent and thephase transfer catalyst for 8 hours or less (e.g., 7 hours or less, 6hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2hours or less, or 1 hour or less). In certain embodiments, step (a) cancomprise contacting the polyvinyl chloride with the cyanation reagentand the phase transfer catalyst for from 10 minutes to 8 hours (e.g.,from 10 minutes to 6 hours, from 10 minutes to 3 hours, from 10 minutesto 2 hours, or from 10 minutes to 1 hour).

The phase transfer catalyst can comprise a salt compound, such as aGroup 15 salt compound (e.g., a quaternary ammonium salt, or aphosphonium salt). Examples of suitable phase transfer catalysts include(but are not limited to) tetrabutyl ammonium bromide, tetrabutylammonium cyanide, tetrabutyl ammonium iodide, tetrabutyl ammoniumnitrate, tetrabutyl ammonium bisulfate, tetrabutyl phosphonium bromide,tetraphenyl phosphonium bromide, or a combination thereof. In someembodiments, the phase transfer catalyst can comprise one of thefollowing:

Examples of suitable cyanation reagents include, for example, cyanidesalts such as NaCN, KCN, trimethylsilyl cyanide, tetraalkylammoniumcyanides, tetraphenylphosphonium cyanides, and combinations thereof. Insome embodiments, the cyanation reagent can comprise NaCN, KCN, or acombination thereof.

In some embodiments, step (b) can be performed at a temperature of from20° C. to 25° C.

In some embodiments, the antimicrobial agent can comprise a hydrazonemoiety or an azide moiety, and step (b) can comprise contacting thecyano-substituted polyvinyl chloride with the antimicrobial agent underconditions effective to covalently bond the antimicrobial agent to thepolyvinyl chloride surface.

In other embodiments, step (b) can comprise (i) converting cyano groupsin the cyano-substituted polyvinyl chloride to 1H-tetrazole moieties,thereby forming a 1H-tetrazole-substituted polyvinyl chloride; and (ii)contacting the 1H-tetrazole-substituted polyvinyl chloride with anantimicrobial agent comprising an electrophilic moiety under conditionseffective to covalently bond the antimicrobial agent to the polyvinylchloride surface. In these embodiments, step (i) can comprise contactingthe cyano-substituted polyvinyl chloride with an azide salt in thepresence of a catalyst, such as ZnBr₂.

In other embodiments, step (b) can comprise (i) converting cyano groupsin the cyano-substituted polyvinyl chloride to 1H-tetrazole moieties,thereby forming a 1H-tetrazole-substituted polyvinyl chloride; (ii)contacting the 1H-tetrazole-substituted polyvinyl chloride with anelectrophile comprising an azide group to form an azide-substitutedpolyvinyl chloride; and (iii) contacting the azide-substituted polyvinylchloride with an antimicrobial agent comprising an alkyne moiety underconditions effective to covalently bond the antimicrobial agent to thepolyvinyl chloride surface. The electrophile comprising the azide groupcan be chosen from, for example, a benzylic halide comprising an azidegroup, an allylic halide comprising an azide group, and a propargylichalide comprising an azide group. In some embodiments, the alkyne moietypresent on the antimicrobial agent can be activated (e.g., activated byring strain, activated by one or more electron withdrawing groups, or acombination thereof). In some of these embodiments, the step (iii) canbe performed without a catalyst. Alternatively, a Cu(I) catalyst may beused to facilitate reaction between the azide on the PVC surface and thealkyne moiety on the antimicrobial agent. In these embodiments, step(iii) can comprise contacting azide-substituted polyvinyl chloride withthe antimicrobial agent comprising the alkyne moiety in the presence ofa Cu(I) catalyst.

In other embodiments, step (b) can comprise (i) converting cyano groupsin the cyano-substituted polyvinyl chloride to 1H-tetrazole moieties,thereby forming a 1H-tetrazole-substituted polyvinyl chloride; (ii)contacting the 1H-tetrazole-substituted polyvinyl chloride with anelectrophile comprising an alkynyl group to form an alkyne-substitutedpolyvinyl chloride; and (iii) contacting the alkyne-substitutedpolyvinyl chloride with an antimicrobial agent comprising an azidemoiety under conditions effective to covalently bond the antimicrobialagent to the polyvinyl chloride surface. The electrophile comprising thealkynyl group can be chosen from, for example, a benzylic halidecomprising an alkynyl group, an allylic halide comprising an alkynylgroup, and a propargylic halide comprising an alkynyl group. In someembodiments, the alkyne group present on the PVC surface can beactivated (e.g., activated by ring strain, activated by one or moreelectron withdrawing groups, or a combination thereof). In some of theseembodiments, the step (iii) can be performed without a catalyst.Alternatively, a Cu(I) catalyst may be used to facilitate reactionbetween the alkyne on the PVC surface and the azide moiety on theantimicrobial agent. In these embodiments, step (iii) can comprisecontacting alkyne-substituted polyvinyl chloride with the antimicrobialagent comprising the azide moiety in the presence of a Cu(I) catalyst.

In some embodiments of the processes described above, the PVC surfacecan comprise a surface of a medical article. In certain embodiments, themedical article can comprise PVC tubing (e.g., endotracheal tubing). Inthe case of PVC tubing, the interior surface of the tubing, the exteriorsurface of the tubing, or both the interior surface of the tubing andthe exterior surface of the tubing can be rendered antimicrobial throughthe processes described above.

As discussed above, these processes can employ mild reaction conditionsthat permit covalent attachment of the antimicrobial agents to a PVCsurface without substantially altering the physiochemical and mechanicalproperties of the article comprising the PVC surface. As a consequence,the processes described herein can be used to impart antimicrobialactivity to a surface of a formed PVC article (e.g., an extruded articlesuch as PVC tubing) without unacceptably compromising the physiochemicaland mechanical properties of the article (e.g., without damaging the PVCtubing).

In some embodiments when the processes described above are performed ona surface of PVC tubing, the process does not significantly impact thehardness of the PVC tubing as measured using the standard methoddescribed in ASTM D-2240-15, entitled Standard Test Method for RubberProperty—Durometer Hardness (2015), which is hereby incorporated byreference in its entirety. For example, in some embodiments, thehardness of the PVC tubing after a process described above is performedis within 20% (e.g., within 15%, within 10%, or within 5%) of thehardness of the PVC tubing before the process described above isperformed, as measured by ASTM D-2240-15.

In some embodiments when the processes described above are performed ona surface of PVC tubing, the process does not significantly impact thetensile strength of the PVC tubing as measured using the standard methoddescribed in ASTM D-638-14, entitled Standard Test Method for TensileProperties of Plastics (2014), which is hereby incorporated by referencein its entirety. For example, in some embodiments, the tensile strengthof the PVC tubing after a process described above is performed is within20% (e.g., within 15%, within 10%, or within 5%) of the tensile strengthof the PVC tubing before the process described above is performed, asmeasured by ASTM D-638-14.

In some embodiments when the processes described above are performed ona surface of PVC tubing, the process does not significantly impact theelongation of the PVC tubing as measured using the standard methoddescribed in ASTM D-638-14, entitled Standard Test Method for TensileProperties of Plastics (2014), which is hereby incorporated by referencein its entirety. For example, in some embodiments, the elongation of thePVC tubing after a process described above is performed is within 20%(e.g., within 15%, within 10%, or within 5%) of the elongation of thePVC tubing before the process described above is performed, as measuredby ASTM D-638-14.

In the processes described above, any suitable antimicrobial agent canbe covalently bound to the PVC surface, provided that (1) theantimicrobial agent includes or can be modified to include a functionalgroup (e.g., an azide, an alkyne, a hydrazone, etc.) which can reactwith a functional group introduced onto the PVC surface to form acovalent bond, and (2) covalent attachment of the antimicrobial agent tothe PVC surface does not eliminate the agent's ability to function as anantimicrobial agent.

In some embodiments, the antimicrobial agent can comprise a conventionalbiocide. A “biocide” as used herein refers to a substance with theability to kill or to inhibit the growth of microorganisms (e.g.,bacteria, fungal cells, protozoa, etc.), such as an antibiotic. Examplesof antibiotics include aminoglycosides, carbacephems (e.g., loracarbef),carbapenems, cephalosporins, glycopeptides (e.g., teicoplanin andvancomycin), macrolides, monobactams (e.g., aztreonam) penicillins,polypeptides (e.g., bacitracin, colistin, polymyxin B), quinolones,sulfonamides, tetracyclines, etc. Antibiotics treat infections by eitherkilling or preventing the growth of microorganisms. Many act to inhibitcell wall synthesis or other vital protein synthesis of themicroorganisms.

Aminoglycosides are commonly used to treat infections caused byGram-negative bacteria such as Escherichia coli and Klebsiella,particularly Pseudomonas aeroginosa. Examples of aminoglycosidesinclude, but are not limited to amikacin, gentamicin, kanamycin,neomycin, netilmicin, streptomycin, tobramycin, and paromomycin.

Carbapenems are broad-specrum antibiotics, and include, but are notlimited to, ertapenem, doripenem, imipenem/cilstatin, and meropenem.

Cephalosporins include, but are not limited to, cefadroxil, cefazolin,cefalotin (cefalothin), cefalexin, cefaclor, cefamandole, cefoxitin,cefprozil, loracarbef, cefuroxime, cefixime, cefdinir, cefditoren,cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten,ceftizoxime, ceftriaxone, cefepime, cefpirome, and ceftobiprole.

Macrolides include, but are not limited to, azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin,troleandomycin, telithromycin and spectinomycin.

Penicillins include, but are not limited to, amoxicillin, ampicillin,azlocillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin,flucloxacillin, mezlocillin, meticillin, nafcillin, oxacillin,penicillin, piperacillin and ticarcillin.

Quinolones include, but are not limited to, ciprofloxacin, enoxacin,gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin,norfloxacin, ofloxacin and trovafloxacin.

Sulfonamides include, but are not limited to, mafenide, prontosil,sulfacetamide, sulfamethizole, sulfanilamide, sulfasalazine,sulfisoxazole, trimethoprim, and co-trimoxazole(trimethoprim-sulfamethoxazole).

Tetracyclines include, but are not limited to, demeclocycline,doxycycline, minocycline, oxytetracycline and tetracycline.

Other antibiotics include arsphenamine, chloramphenicol, clindamycin,lincomycin, ethambutol, fosfomycin, fusidic acid, furazolidone,isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin,platensimycin, pyrazinamide, quinupristin/dalfopristin, rifampin(rifampicin), tinidazole, etc.

In some embodiments of the processes described above, the antimicrobialagent can comprise an antimicrobial polymer. Antimicrobial polymers areknown in the art, and include, for example, polylactams, polyaminoacids, polymers containing tertiary and/or quaternary ammonium groups,and polymers containing pyridinium sidechains. In certain embodiments,the antimicrobial polymer can comprise a polycationic polymer thatincludes a plurality of positively charged centers, each of which isformed by condensation of cyclic bis-electrophile, such as a9-thia/aza/selenabicyclo[3.3.1]nonyl electrophile. Such polycationicpolymers are described in more detail below.

Articles

Antimicrobial articles are also provided herein. The antimicrobialarticles can comprise a surface (e.g., a surface of a substrate), and apolycationic polymer derived from the condensation of a cyclicbis-electrophile and a polynucleophilic monomer immobilized on thesurface. The cyclic bis-electrophile can be defined by Formula II below

where each dotted line represents a cyclic moiety; LG represents aleaving group; Z represents S, Se, or NR³; and R³ represents alkyl,alkenyl, alkynyl, alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl,cycloalkyl alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl,peptidyl, polyamino, or polyalkyleneoxy. In some embodiments, the cyclicbis-electrophile can be defined by Formula IIA, Formula IIB, or FormulaIIC below

wherein LG and Z are as defined above with respect to Formula II. Thepolynucleophilic monomer can be defined by Formula III below

wherein A represents a heterocyclic ring comprising a tertiary nitrogenatom; Y is absent or represents a linking group; and R¹ is absent, orrepresents halogen, hydroxy, amino, cyano, azido, hydrazone, alkyl,alkenyl, alkynyl, alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl,cycloalkyl alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, dialkylamino,alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,peptidyl, polyamino, or polyalkyleneoxy.

Polycationic polymers of this type are discussed in more detail below.The polycationic polymers can be bound to the surface covalently ornon-covalently using any suitable strategy known in the art.

The surface can be of any composition, such as a ceramic, glass, metal,wood, or polymer. In certain embodiments, the surface comprises apolymer surface (e.g., a polyvinyl chloride surface).

The article can comprise any of the substrates described below. In someembodiments, the article can comprise a medical device. Examples ofmedical device include pins, screws, plates, ventriculoatrial shunts,ventriculoperitoneal shunts, dialysis shunts, heart valves, pacemakers,infusion pumps, vascular grafting prostheses, stents, sutures, surgicalmeshes, replacement prostheses, breast implants, tissue expanders,contact lenses, stoma appliances, artificial larynx, endotracheal tubes,tracheal tubes, gastrostomy tubes, biliary drainage tubes, biliarystents, catheters, bandages, adhesive tapes, and clear plastic adherentsheets. In certain embodiments, the article can comprise polyvinylchloride tubing.

Methods

Also provided are methods for controlling biofilm formation on asubstrate. Methods can comprise covalently bonding a polycationicpolymer to the substrate in an amount effective to inhibit biofilmformation.

The polycationic polymer can be derived from the condensation of acyclic bis-electrophile and a polynucleophilic monomer immobilized onthe surface. The cyclic bis-electrophile can be defined by Formula IIbelow

where each dotted line represents a cyclic moiety; LG represents aleaving group; Z represents S, Se, or NR³; and R³ represents alkyl,alkenyl, alkynyl, alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl,cycloalkyl alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl,peptidyl, polyamino, or polyalkyleneoxy. In some embodiments, the cyclicbis-electrophile can be defined by Formula IIA, Formula IIB, or FormulaIIC below

wherein LG and Z are as defined above with respect to Formula II. Thepolynucleophilic monomer can be defined by Formula III below

wherein A represents a heterocyclic ring comprising a tertiary nitrogenatom; Y is absent or represents a linking group; and R¹ is absent, orrepresents halogen, hydroxy, amino, cyano, azido, hydrazone, alkyl,alkenyl, alkynyl, alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl,cycloalkyl alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, dialkylamino,alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,peptidyl, polyamino, or polyalkyleneoxy.

Polycationic polymers of this type are discussed in more detail below.The polycationic polymers can be bound to the surface covalently ornon-covalently using any suitable strategy known in the art.

The substrate can be of any composition, such as a ceramic, glass,metal, wood, or polymer. In certain embodiments, the substrate comprisesa polymer substrate (e.g., a polyvinyl chloride substrate).

The substrate can comprise any of the substrates described below. Insome embodiments, the substrate can comprise a medical device. Examplesof medical device include pins, screws, plates, ventriculoatrial shunts,ventriculoperitoneal shunts, dialysis shunts, heart valves, pacemakers,infusion pumps, vascular grafting prostheses, stents, sutures, surgicalmeshes, replacement prostheses, breast implants, tissue expanders,contact lenses, stoma appliances, artificial larynx, endotracheal tubes,tracheal tubes, gastrostomy tubes, biliary drainage tubes, biliarystents, catheters, bandages, adhesive tapes, and clear plastic adherentsheets. In certain embodiments, the article can comprise polyvinylchloride tubing.

The biofilm can comprise Gram-positive bacteria or Gram-negativebacteria. In some embodiments, the biofilm can comprise Gram-positivebacteria. Examples of Gram-positive bacteria affected by compoundsdescribed herein include, but are not limited to, bacteria of the generaListeria, Staphylococcus, Streptococcus, Bacillus, Corynebacterium,Peptostreptococcus, and Clostridium. For example, the bacteria caninclude Listeria monocytogenes, Staphylococcus aureus, Streptococcuspyogenes, Streptococcus pneumoniae, Bacillus cereus, Bacillus anthracis,Clostridium botulinum, Clostridium perfringens, Clostridium difficile,Clostridium tetani, Corynebacterium diphtheriae, Corynebacteruimulcerans, and Peptostreptococcus anaerobius. Other examples ofGram-positive bacteria include, for example, bacteria of the generaActinomyces, Propionibacterium, Nocardia and Streptomyces.

In some embodiments, the biofilm can comprise Gram-negative bacteria.Examples of Gram-positive bacteria affected by compounds describedherein include, but are not limited to, bacteria of the generaEscherichia, Salmonella, Vibrio, Helicobacter, Pseudomonas, Bordetella,Vibrio, Haemophilus, Halomonas, and Acinetobacter. For example, thebacteria can include Pseudomonas aeuroginosa, Bordetella pertussis,Vibrio vulnificus, Haemophilus influenzae, Halomonas pacifica, andAcinetobacter baumannii. Other examples of Gram-negative bacteriainclude, for example, bacteria of the genera Klebsiella, Proteus,Neisseria, Helicobacter, Brucella, Legionella, Campylobacter,Francisella, Pasteurella, Yersinia, Bartonella, Bacteroides,Streptobacillus, Spirillum, Moraxella and Shigella.

Substrates and Surfaces

The term “substrate” as used herein refers to bases on which anorganism, such as those commonly found in biofilms, may live. The term“substrate,” as used herein, refers to any substrate, whether in anindustrial or a medical setting, that provides or can provide aninterface between an object and a fluid, permitting at leastintermittent contact between the object and the fluid. A substrate, asunderstood herein, further provides a plane whose mechanical structure,without further treatment, is compatible with the adherence ofmicroorganisms. Substrates compatible with biofilm formation may benatural or synthetic, and may be smooth or irregular. Fluids contactingthe substrates can be stagnant or flowing, and can flow intermittentlyor continuously, with laminar or turbulent or mixed flows. A substrateupon which a biofilm forms can be dry at times with sporadic fluidcontact, or can have any degree of fluid exposure including totalimmersion. Fluid contact with the substrate can take place via aerosolsor other means for air-borne fluid transmission.

Biofilm formation with health implications can involve those substratesin all health-related environments, including substrates found inmedical environments and those substrates in industrial or residentialenvironments that are involved in those functions essential to humanwell being, for example, nutrition, sanitation and the prevention ofdisease. Substrates found in medical environments include the inner andouter aspects of various instruments and devices, whether disposable orintended for repeated uses. Examples include the entire spectrum ofarticles adapted for medical use, including scalpels, needles, scissorsand other devices used in invasive surgical, therapeutic or diagnosticprocedures; implantable medical devices, including artificial bloodvessels, catheters and other devices for the removal or delivery offluids to patients, artificial hearts, artificial kidneys, orthopedicpins, plates and implants; catheters and other tubes (includingurological and biliary tubes, endotracheal tubes, peripherablyinsertable central venous catheters, dialysis catheters, long termtunneled central venous catheters, peripheral venous catheters, shortterm central venous catheters, arterial catheters, pulmonary catheters,Swan-Ganz catheters, urinary catheters, peritoneal catheters), urinarydevices (including long term urinary devices, tissue bonding urinarydevices, artificial urinary sphincters, urinary dilators), shunts(including ventricular or arterio-venous shunts); prostheses (includingbreast implants, penile prostheses, vascular grafting prostheses, heartvalves, artificial joints, artificial larynxes, otological implants),vascular catheter ports, wound drain tubes, hydrocephalus shunts,pacemakers and implantable defibrillators, and the like. Other exampleswill be readily apparent to practitioners in these arts. Substratesfound in the medical environment also include the inner and outeraspects of pieces of medical equipment, medical gear worn or carried bypersonnel in the health care setting. Such substrates can includecounter tops and fixtures in areas used for medical procedures or forpreparing medical apparatus, tubes and canisters used in respiratorytreatments, including the administration of oxygen, of solubilized drugsin nebulizers and of anesthetic agents. Also included are thosesubstrates intended as biological barriers to infectious organisms inmedical settings, such as gloves, aprons and faceshields. Commonly usedmaterials for biological barriers may be latex-based or non-latex based.Vinyl is commonly used as a material for non-latex surgical gloves.Other such substrates can include handles and cables for medical ordental equipment not intended to be sterile. Additionally, suchsubstrates can include those non-sterile external substrates of tubesand other apparatus found in areas where blood or body fluids or otherhazardous biomaterials are commonly encountered.

Substrates in contact with liquids are particularly prone to biofilmformation. As an example, those reservoirs and tubes used for deliveringhumidified oxygen to patients can bear biofilms inhabited by infectiousagents. Dental unit waterlines similarly can bear biofilms on theirsubstrates, providing a reservoir for continuing contamination of thesystem of flowing an aerosolized water used in dentistry. Sprays,aerosols and nebulizers are highly effective in disseminating biofilmfragments to a potential host or to another environmental site. It isespecially important to health to prevent biofilm formation on thosesubstrates from where biofilm fragments can be carried away by sprays,aerosols or nebulizers contacting the substrate.

Other substrates related to health include the inner and outer aspectsof those articles involved in water purification, water storage andwater delivery, and articles involved in food processing. Substratesrelated to health can also include the inner and outer aspects of thosehousehold articles involved in providing for nutrition, sanitation ordisease prevention. Examples can include food processing equipment forhome use, materials for infant care, tampons and toilet bowls.

Substrates can be smooth or porous, soft or hard. Substrates can includea drainpipe, glaze ceramic, porcelain, glass, metal, wood, chrome,plastic, vinyl, Formica® brand laminate, or any other material that mayregularly come in contact with an aqueous solution in which biofilms mayform and grow. The substrate can be a substrate commonly found onhousehold items such as shower curtains or liners, upholstery, laundry,and carpeting.

Another substrate on which biofilm preventing, removing or inhibiting isimportant is that of a ship hull. Biofilms, such as those of Halomonaspacifica, promote the corrosion of the hull of ships and also increasethe roughness of the hull, increasing the drag on the ship and therebyincreasing fuel costs. The biofilm can also promote the attachment oflarger living structures such as barnacles on the ship hull. Fuel canaccount for half of the cost of marine shipping, and the loss in fuelefficiency due to biofilm formation is substantial.

Substrates on which biofilms can adhere include those of livingorganisms, as in the case of humans with chronic infections caused bybiofilms, as discussed above. Biofilms can also form on the substratesof food contact surfaces, such as those used for processing seafood, andalso on food products themselves. Examples of seafood products that mayhave biofilm contamination include oysters. Human infections caused bythe ingestion of raw oysters has been linked to Vibrio vulnificusbacterium. Vibrio bacteria attach to algae and plankton in the water andtransfer to the oysters and fish that feed on these organisms.

Other examples of substrates or devices on which biofilms can adhere canbe found in U.S. Pat. Nos. 5,814,668 and 7,087,661; and U.S. Pat.Application Publication Nos. 2006/0228384 and 2006/0018945, each ofwhich is incorporated herein by reference in its entirety.

The term “surface” as used herein refers, can refer to a surface of anyof the substrates described above.

Polycationic Polymers

Also provided are antimicrobial polycationic polymers that can be usedin conjunction with the processes, articles, and methods describedabove. The polycationic polymers can include a plurality of positivelycharged centers, each of which is formed by condensation of cyclicbis-electrophile, such as a 9-thia/aza/selenabicyclo[3.3.1]nonylelectrophile, with a nucleophile. The resulting polycationic polymerscan efficiently bind nucleic acids. The polycations can also exhibitproperties of cytotoxicity and DNA transfection with interestingstructure-activity characteristics.

In some cases, the polycationic polymer can be formed by thecondensation of cyclic bis-electrophile (e.g., a9-thia/aza/selenabicyclo[3.3.1]nonyl dielectrophile) with apolynucleophiles (e.g., a dinucleophiles). Each bond-forming event inthis condensation polymerization process generates a positively chargedcenter within the polymer backbone. In these systems, the length of thepolymer chain was found to be dependent on the reactivity of theelectrophile, which can be readily tuned through variation of theleaving group β to sulfur/nitrogen/selenium atom present in the cyclicbis-electrophile. These polycations can also be biodegradable.Degradation of the polycations can occur via hydrolysis, with the rateof hydrolysis dependent on the leaving-group ability of the nucleophile(which correlates roughly with the pK_(a) of the nucleophile's conjugateacid). Thus, the degradation rates of the polycations can be tunedthrough selection of an appropriate nucleophile. Polymer decompositionoccurs simultaneously throughout the length of the chains, rather thanfrom the ends; the decomposition products can be only minimally toxic tomammalian cells.

In some embodiments, the polycationic polymer can be derived from thecondensation of a cyclic bis-electrophile and a polynucleophilicmonomer. The cyclic bis-electrophile can be defined by Formula II below

where each dotted line represents a cyclic moiety; LG represents aleaving group; Z represents S, Se, or NR³; and R³ represents alkyl,alkenyl, alkynyl, alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl,cycloalkyl alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl,peptidyl, polyamino, or polyalkyleneoxy. In some embodiments, the cyclicbis-electrophile can be defined by Formula IIA, Formula IIB, or FormulaIIC below

wherein LG and Z are as defined above with respect to Formula II. Thepolynucleophilic monomer can be defined by Formula III below

wherein A represents a heterocyclic ring comprising a tertiary nitrogenatom; Y is absent or represents a linking group; and R¹ is absent, orrepresents halogen, hydroxy, amino, cyano, azido, hydrazone, alkyl,alkenyl, alkynyl, alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl,cycloalkyl alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, dialkylamino,alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,peptidyl, polyamino, or polyalkyleneoxy.

In some embodiments, the polycationic polymer can be derived from thecondensation of a single species of cyclic bis-electrophile defined byFormula II and a single species of polynucleophilic monomer defined byFormula III. In other embodiments, the polycationic polymer can bederived from the condensation of a single species of cyclicbis-electrophile defined by Formula II and two or more different speciesof polynucleophilic monomer defined by Formula III. In otherembodiments, the polycationic polymer can be derived from thecondensation of two or more different species of cyclic bis-electrophiledefined by Formula II and a single species of polynucleophilic monomerdefined by Formula III. In other embodiments, the polycationic polymercan be derived from the condensation of two or more different species ofcyclic bis-electrophile defined by Formula II and two or more differentspecies of polynucleophilic monomer defined by Formula III.

In some embodiments of Formula II, Z can be S. In other embodiments, Zcan be NR³.

In some embodiments of Formula II, Z can be NR³, and R³ can comprise acationic group. For example, in certain embodiments, the cationic groupcan comprise a cationic polyamino group (e.g., a polyethyleneiminesegment). In other embodiments, the cationic group can comprise acationic peptidyl group. In other embodiments, the cationic group cancomprise a moiety (e.g., an alkyl group or an alkylaryl group)substituted with a cationic substituent, such as an ammonium group(e.g., a tetraalkylammonium group, a aryltrialkylammonium group, adiaryldialkylammonium group, or a triarylalkylammonium group) or apyridinium group.

In some embodiments of Formula II, Z can be NR³, and R³ can comprise ahydrophobic group. For example, in certain embodiments, Z can be NR³,and R³ can comprise an aryl group or an alkylaryl group.

In some embodiments of Formula II, Z can be NR³, and R³ can comprise ahydrophilic group. For example, in certain embodiments, Z can be NR³,and R³ can comprise a hydrophilic polyalkyleneoxy group (e.g., apolyethylene oxide segment).

In some embodiments of Formula II, Z can be NR³, and R³ can comprise areactive functional group. Reactive functional groups include moietiesthat can participate in a reaction with another moiety, which may alsobe a reactive functional group, to form a covalent bond. Examples ofreactive functional groups include, but are not limited to, olefins,acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes,ketones, carboxylic acids, esters, amides, cyanates, isocyanates,thiocyanates, isothiocyanates, amines, hydrazines, hydrazones,hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides,disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids,acetals, ketals, anhydrides, sulfates, sulfenic acids isonitriles,amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamicacids thiohydroxamic acids, allenes, ortho esters, sulfites, enamines,ynamines, ureas, pseudoureas, semicarbazides, carbodiimides, carbamates,imines, azides, azo compounds, azoxy compounds, and nitroso compounds.Reactive functional groups also include those used to preparebioconjugates, e.g., N-hydroxysuccinimide esters, maleimides and thelike. Methods to prepare each of these functional groups are well knownin the art and their application to or modification for a particularpurpose is within the ability of one of skill in the art. See, forexample, Sandler and Karo, eds. Organic Functional Group Preparations,Academic Press, San Diego, 1989. For example, in certain embodiments, Zcan be NR³, and R³ can comprise an alkynyl group or an azido group. Inother embodiments, Z can be NR³, and R³ can comprise a hydrazone group.

LG can represent any suitable leaving group. Leaving groups are wellknown in the art, and include substituent present within a chemicalcompound that can be readily displaced. Examples of leaving groupsinclude halides (chloride, bromide, and iodide), triflates (OTf), boronmoieties including boronic acids and trihaloborate salts such astrifluoroborate salts (BF₃), diazonium salts (N₂ ⁺), tosylates (0Ts) andother sulfonic esters, mesylates (OMs), nitrates (—ONO₂), and phosphates(—OPO(OH)₂). In some examples, LG can be a halide (e.g., chloride,bromide, or iodide). In other examples, LG can be triflate. In otherexamples, LG can be nitrate.

A can be any heterocyclic ring comprising a tertiary nitrogen atom. Insome embodiments, A can be a heteroaromatic ring. In some embodiments, Acan comprise one or more additional heteroatoms in addition to thetertiary nitrogen atom (e.g., one or more additional nitrogen atoms, oneor more oxygen atoms, one or more sulfur atoms, or a combinationthereof). For example, A can be chosen from pyridine, imidazole,pyrazole, triazole, tetrazole, oxazole, isoxazole, furazan,isothioazole, and thiazole. In certain embodiments, A can represent apyridine ring.

In some embodiments, Y can be absent (i.e., the two heterocyclic ringscan be bound directly to one another). In other embodiments, Y ispresent. When present, the linking group can be any suitable group ormoiety which is at minimum bivalent, and connects the heterocyclic ringsin the nucleophile. The linking group can be composed of any assembly ofatoms, including oligomeric and polymeric chains. In some cases, thetotal number of atoms in the linking group can be from 3 to 200 atoms(e.g., from 3 to 150 atoms, from 3 to 100 atoms, from 3 and 50 atoms,from 3 to 25 atoms, from 3 to 15 atoms, or from 3 to 10 atoms). In someembodiments, the linking group can be, for example, an alkyl, alkoxy,alkylaryl, alkylheteroaryl, alkylcycloalkyl, alkylheterocycloalkyl,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, dialkylamino,alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,or polyamino group. In some embodiments, the linking group can comprisesone of the groups above joined to each of the heterocyclic rings by afunctional group. Examples of suitable functional groups include, forexample, secondary amides (—CONH—), tertiary amides (—CONR—), secondarycarbamates (—OCONH—; —NHCOO—), tertiary carbamates (—OCONR—; —NRCOO—),ureas (—NHCONH—; —NRCONH—; —NHCONR—, or —NRCONR—), carbinols (—CHOH—,—CROH—), ethers (—O—), and esters (—COO—, —CH₂O₂C—, CHRO₂C—), wherein Ris an alkyl group, an aryl group, or a heterocyclic group. For example,in some embodiments, the linking group can comprise an alkyl group(e.g., a C₁-C₁₂ alkyl group, a C₁-C₈ alkyl group, or a C₁-C₆ alkylgroup) bound to each heterocyclic ring via an ester (—COO—, —CH₂O₂C—,CHRO₂C—), a secondary amide (—CONH—), or a tertiary amide (—CONR—),wherein R is an alkyl group, an aryl group, or a heterocyclic group. Incertain embodiments, Y can be chosen from one of the following:

where m is an integer from 1 to 12.

In some embodiments, R¹ can be absent. In other embodiments, R¹ can bepresent. When present, R¹ can comprise a halogen, hydroxy, amino, cyano,azido, hydrazone, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl,alkylaryl, alkylheteroaryl, cycloalkyl alkylcycloalkyl,heterocycloalkyl, alkylheterocycloalkyl, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, dialkylamino, alkylcarbonyl, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, peptidyl, polyamino, orpolyalkyleneoxy group.

In some embodiments, R¹ can comprise a cationic group. In certainembodiments, the cationic group can comprise a cationic polyamino group(e.g., a polyethyleneimine segment). In other embodiments, the cationicgroup can comprise a cationic peptidyl group. In other embodiments, thecationic group can comprise a moiety (e.g., an alkyl group or analkylaryl group) substituted with a cationic substituent, such as anammonium group (e.g., a tetraalkylammonium group, a aryltrialkylammoniumgroup, a diaryldialkylammonium group, or a triarylalkylammonium group)or a pyridinium group.

In some embodiments, R¹ can comprise a hydrophobic group. For example,in certain embodiments, R¹ can comprise an aryl group or an alkylarylgroup. In some embodiments, R¹ can comprise a hydrophilic group. Forexample, in certain embodiments, R¹ can comprise a hydrophilicpolyalkyleneoxy group (e.g., a polyethylene oxide segment).

In some embodiments, R¹ can comprise a reactive functional group, suchas an olefin, acetylene, alcohol, phenol, ether, oxide, halide,aldehyde, ketone, carboxylic acid, ester, amide, cyanate, isocyanate,thiocyanate, isothiocyanate, amine, hydrazine, hydrazone, hydrazide,diazo, diazonium, nitro, nitrile, mercaptan, sulfide, disulfide,sulfoxide, sulfone, sulfonic acid, sulfinic acid, acetal, ketal,anhydride, sulfate, sulfenic acid isonitrile, amidine, imide, imidate,nitrone, hydroxylamine, oxime, hydroxamic acid, thiohydroxamic acid,allene, ortho ester, sulfite, enamine, ynamine, urea, pseudourea,semicarbazide, carbodiimide, carbamate, imine, azide, azo group, azoxygroup, nitroso group, N-hydroxysuccinimide ester, or maleimides. Forexample, in certain embodiments, R¹ can comprise an alkynyl group or anazido group. In other embodiments, R¹ can comprise a hydrazone group.

In some embodiments, the polycationic polymer polymer can be derivedfrom the condensation of one or more cyclic bis-electrophiles defined byFormula II, one or more polynucleophilic monomers defined by FormulaIII, and one or more polynucleophilic monomers defined by Formula IVbelow

wherein A represents a heterocyclic ring comprising a tertiary nitrogenatom; Y is absent or represents a linking group; and R¹ is absent, orrepresents halogen, hydroxy, amino, cyano, azido, hydrazone, alkyl,alkenyl, alkynyl, alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl,cycloalkyl alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, dialkylamino,alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,peptidyl, polyamino, or polyalkyleneoxy.

In Formula IV, A can be any heterocyclic ring comprising a tertiarynitrogen atom. In some embodiments, A can be a heteroaromatic ring. Insome embodiments, A can comprise one or more additional heteroatoms inaddition to the tertiary nitrogen atom (e.g., one or more additionalnitrogen atoms, one or more oxygen atoms, one or more sulfur atoms, or acombination thereof). For example, A can be chosen from pyridine,imidazole, pyrazole, triazole, tetrazole, oxazole, isoxazole, furazan,isothioazole, and thiazole. In certain embodiments, A can represent apyridine ring.

In some embodiments of Formula IV, Y can be absent (i.e., the twoheterocyclic rings can be bound directly to one another). In otherembodiments, Y is present. When present, the linking group can be anysuitable group or moiety which is at minimum bivalent, and connects theheterocyclic rings in the nucleophile. The linking group can be composedof any assembly of atoms, including oligomeric and polymeric chains. Insome cases, the total number of atoms in the linking group can be from 3to 200 atoms (e.g., from 3 to 150 atoms, from 3 to 100 atoms, from 3 and50 atoms, from 3 to 25 atoms, from 3 to 15 atoms, or from 3 to 10atoms). In some embodiments, the linking group can be, for example, analkyl, alkoxy, alkylaryl, alkylheteroaryl, alkylcycloalkyl,alkylheterocycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, dialkylamino, alkylcarbonyl, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, or polyamino group. In someembodiments, the linking group can comprises one of the groups abovejoined to each of the heterocyclic rings by a functional group. Examplesof suitable functional groups include, for example, secondary amides(—CONH—), tertiary amides (—CONR—), secondary carbamates (—OCONH—;—NHCOO—), tertiary carbamates (—OCONR—; —NRCOO—), ureas (—NHCONH—;—NRCONH—; —NHCONR—, or —NRCONR—), carbinols (—CHOH—, —CROH—), ethers(—O—), and esters (—COO—, —CH₂O₂C—, CHRO₂C—), wherein R is an alkylgroup, an aryl group, or a heterocyclic group. For example, in someembodiments, the linking group can comprise an alkyl group (e.g., aC₁-C₁₂ alkyl group, a C₁-C₈ alkyl group, or a C₁-C₆ alkyl group) boundto each heterocyclic ring via an ester (—COO—, —CH₂O₂C—, CHRO₂C—), asecondary amide (—CONH—), or a tertiary amide (—CONR—), wherein R is analkyl group, an aryl group, or a heterocyclic group.

In some embodiments of Formula IV, R¹ can be absent. In otherembodiments, R¹ can be present. When present, R¹ can comprise a halogen,hydroxy, amino, cyano, azido, hydrazone, alkyl, alkenyl, alkynyl,alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl, cycloalkylalkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, dialkylamino, alkylcarbonyl,alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, peptidyl,polyamino, or polyalkyleneoxy group.

In some embodiments of Formula IV, R¹ can comprise a cationic group. Incertain embodiments, the cationic group can comprise a cationicpolyamino group (e.g., a polyethyleneimine segment). In otherembodiments, the cationic group can comprise a cationic peptidyl group.In other embodiments, the cationic group can comprise a moiety (e.g., analkyl group or an alkylaryl group) substituted with a cationicsubstituent, such as an ammonium group (e.g., a tetraalkylammoniumgroup, an aryltrialkylammonium group, a diaryldialkylammonium group, ora triarylalkylammonium group) or a pyridinium group.

In some embodiments of Formula IV, R¹ can comprise a hydrophobic group.For example, in certain embodiments, R¹ can comprise an aryl group or analkylaryl group. In some embodiments, R¹ can comprise a hydrophilicgroup. For example, in certain embodiments, R¹ can comprise ahydrophilic polyalkyleneoxy group (e.g., a polyethylene oxide segment).

In some embodiments of Formula IV, R¹ can comprise a reactive functionalgroup, such as an olefin, acetylene, alcohol, phenol, ether, oxide,halide, aldehyde, ketone, carboxylic acid, ester, amide, cyanate,isocyanate, thiocyanate, isothiocyanate, amine, hydrazine, hydrazone,hydrazide, diazo, diazonium, nitro, nitrile, mercaptan, sulfide,disulfide, sulfoxide, sulfone, sulfonic acid, sulfinic acid, acetal,ketal, anhydride, sulfate, sulfenic acid isonitrile, amidine, imide,imidate, nitrone, hydroxylamine, oxime, hydroxamic acid, thiohydroxamicacid, allene, ortho ester, sulfite, enamine, ynamine, urea, pseudourea,semicarbazide, carbodiimide, carbamate, imine, azide, azo group, azoxygroup, nitroso group, N-hydroxysuccinimide ester, or maleimides. Forexample, in certain embodiments, R¹ can comprise an alkynyl group or anazido group. In other embodiments, R¹ can comprise a hydrazone group.

In some embodiments, the polycation polymer can be a polymer defined byFormula I below

wherein Z represents, individually for each occurrence within thepolymer, S, Se, or NR³; A represents, individually for each occurrencewithin the polymer, a heterocyclic ring comprising a cationic nitrogencenter; X represents, individually for each occurrence within thepolymer, an anion; Y is absent, or represents, individually for eachoccurrence within the polymer, a linking group; R¹ is absent, orrepresents, individually for each occurrence within the polymer,halogen, hydroxy, amino, cyano, azido, hydrazone, alkyl, alkenyl,alkynyl, alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl,cycloalkyl alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, dialkylamino,alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,peptidyl, polyamino, or polyalkyleneoxy; R² is absent, or represents,individually for each occurrence within the polymer, alkyl, alkenyl,alkynyl, alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl,cycloalkyl alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl,peptidyl, polyamino, or polyalkyleneoxy; R³ represents, individually foreach occurrence within the polymer, alkyl, alkenyl, alkynyl, alkoxy,aryl, heteroaryl, alkylaryl, alkylheteroaryl, cycloalkylalkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl, peptidyl,polyamino, or polyalkyleneoxy; and n is an integer from 2 to 400.

In Formula I, A can be any heterocyclic ring comprising a tertiarynitrogen atom. In some embodiments, A can be a heteroaromatic ring. Insome embodiments, A can comprise one or more additional heteroatoms inaddition to the tertiary nitrogen atom (e.g., one or more additionalnitrogen atoms, one or more oxygen atoms, one or more sulfur atoms, or acombination thereof). For example, A can be chosen from pyridine,imidazole, pyrazole, triazole, tetrazole, oxazole, isoxazole, furazan,isothioazole, and thiazole. In certain embodiments, A can represent apyridine ring. In these embodiments, the polycation can be defined byFormula IA below

In some embodiments of Formula I and Formula IA, Z can be S. In otherembodiments, Z can be NR³.

In some embodiments of Formula I and Formula IA, Z can be NR³, and R³can comprise a cationic group. For example, in certain embodiments, thecationic group can comprise a cationic polyamino group (e.g., apolyethyleneimine segment). In other embodiments, the cationic group cancomprise a cationic peptidyl group. In other embodiments, the cationicgroup can comprise a moiety (e.g., an alkyl group or an alkylaryl group)substituted with a cationic substituent, such as an ammonium group(e.g., a tetraalkylammonium group, an aryltrialkylammonium group, adiaryldialkylammonium group, or a triarylalkylammonium group) or apyridinium group.

In some embodiments of Formula I and Formula IA, Z can be NR³, and R³can comprise a hydrophobic group. For example, in certain embodiments, Zcan be NR³, and R³ can comprise an aryl group or an alkylaryl group.

In some embodiments of Formula I and Formula IA, Z can be NR³, and R³can comprise a hydrophilic group. For example, in certain embodiments, Zcan be NR³, and R³ can comprise a hydrophilic polyalkyleneoxy group(e.g., a polyethylene oxide segment).

In some embodiments of Formula I and Formula IA, Z can be NR³, and R³can comprise a reactive functional group, such as an olefin, acetylene,alcohol, phenol, ether, oxide, halide, aldehyde, ketone, carboxylicacid, ester, amide, cyanate, isocyanate, thiocyanate, isothiocyanate,amine, hydrazine, hydrazone, hydrazide, diazo, diazonium, nitro,nitrile, mercaptan, sulfide, disulfide, sulfoxide, sulfone, sulfonicacid, sulfinic acid, acetal, ketal, anhydride, sulfate, sulfenic acidisonitrile, amidine, imide, imidate, nitrone, hydroxylamine, oxime,hydroxamic acid, thiohydroxamic acid, allene, ortho ester, sulfite,enamine, ynamine, urea, pseudourea, semicarbazide, carbodiimide,carbamate, imine, azide, azo group, azoxy group, nitroso group,N-hydroxysuccinimide ester, or maleimides. For example, in certainembodiments, R³ can comprise an alkynyl group or an azido group. Inother embodiments, R³ can comprise a hydrazone group.

In Formula I and Formula IA, X can comprise any suitable, stable anion.By way of example, X can be chosen from chloride, bromide, iodide,nitrate, sulfate, triflate, borate, and phosphate.

In some embodiments of Formula I and Formula IA, Y can be absent (i.e.,the two heterocyclic rings can be bound directly to one another). Inother embodiments, Y is present. When present, the linking group can beany suitable group or moiety which is at minimum bivalent, and connectsthe heterocyclic rings in the nucleophile. The linking group can becomposed of any assembly of atoms, including oligomeric and polymericchains. In some cases, the total number of atoms in the linking groupcan be from 3 to 200 atoms (e.g., from 3 to 150 atoms, from 3 to 100atoms, from 3 and 50 atoms, from 3 to 25 atoms, from 3 to 15 atoms, orfrom 3 to 10 atoms). In some embodiments, the linking group can be, forexample, an alkyl, alkoxy, alkylaryl, alkylheteroaryl, alkylcycloalkyl,alkylheterocycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, dialkylamino, alkylcarbonyl, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, or polyamino group. In someembodiments, the linking group can comprises one of the groups abovejoined to each of the heterocyclic rings by a functional group. Examplesof suitable functional groups include, for example, secondary amides(—CONH—), tertiary amides (—CONR—), secondary carbamates (—OCONH—;—NHCOO—), tertiary carbamates (—OCONR—; —NRCOO—), ureas (—NHCONH—;—NRCONH—; —NHCONR—, or —NRCONR—), carbinols (—CHOH—, —CROH—), ethers(—O—), and esters (—COO—, —CH₂O₂C—, CHRO₂C—), wherein R is an alkylgroup, an aryl group, or a heterocyclic group. For example, in someembodiments, the linking group can comprise an alkyl group (e.g., aC₁-C₁₂ alkyl group, a C₁-C₈ alkyl group, or a C₁-C₆ alkyl group) boundto each heterocyclic ring via an ester (—COO—, —CH₂O₂C—, CHRO₂C—), asecondary amide (—CONH—), or a tertiary amide (—CONR—), wherein R is analkyl group, an aryl group, or a heterocyclic group. In certainembodiments, Y can be chosen from one of the following:

where m is an integer from 1 to 12.

In some embodiments of Formula I and Formula IA, R¹ can be absent. Inother embodiments, R¹ can be present. When present, R¹ can comprise ahalogen, hydroxy, amino, cyano, azido, hydrazone, alkyl, alkenyl,alkynyl, alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl,cycloalkyl alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, dialkylamino,alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,peptidyl, polyamino, or polyalkyleneoxy group.

In some embodiments of Formula I and Formula IA, R¹ can comprise acationic group. In certain embodiments, the cationic group can comprisea cationic polyamino group (e.g., a polyethyleneimine segment). In otherembodiments, the cationic group can comprise a cationic peptidyl group.In other embodiments, the cationic group can comprise a moiety (e.g., analkyl group or an alkylaryl group) substituted with a cationicsubstituent, such as an ammonium group (e.g., a tetraalkylammoniumgroup, a aryltrialkylammonium group, a diaryldialkylammonium group, or atriarylalkylammonium group) or a pyridinium group.

In some embodiments of Formula I and Formula IA, R¹ can comprise ahydrophobic group. For example, in certain embodiments, R¹ can comprisean aryl group or an alkylaryl group. In some embodiments, R¹ cancomprise a hydrophilic group. For example, in certain embodiments, R¹can comprise a hydrophilic polyalkyleneoxy group (e.g., a polyethyleneoxide segment).

In some embodiments of Formula I and Formula IA, R¹ can comprise areactive functional group, such as an olefin, acetylene, alcohol,phenol, ether, oxide, halide, aldehyde, ketone, carboxylic acid, ester,amide, cyanate, isocyanate, thiocyanate, isothiocyanate, amine,hydrazine, hydrazone, hydrazide, diazo, diazonium, nitro, nitrile,mercaptan, sulfide, disulfide, sulfoxide, sulfone, sulfonic acid,sulfinic acid, acetal, ketal, anhydride, sulfate, sulfenic acidisonitrile, amidine, imide, imidate, nitrone, hydroxylamine, oxime,hydroxamic acid, thiohydroxamic acid, allene, ortho ester, sulfite,enamine, ynamine, urea, pseudourea, semicarbazide, carbodiimide,carbamate, imine, azide, azo group, azoxy group, nitroso group,N-hydroxysuccinimide ester, or maleimides. For example, in certainembodiments, R¹ can comprise an alkynyl group or an azido group. Inother embodiments, R¹ can comprise a hydrazone group.

In some embodiments of Formula I and Formula IA, R² can be absent. Inother embodiments, R² can be present. When present, R² can comprise analkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, alkylaryl,alkylheteroaryl, cycloalkyl alkylcycloalkyl, heterocycloalkyl,alkylheterocycloalkyl, peptidyl, polyamino, or polyalkyleneoxy

In some embodiments of Formula I and Formula IA, R² can comprise acationic group. In certain embodiments, the cationic group can comprisea cationic polyamino group (e.g., a polyethyleneimine segment). In otherembodiments, the cationic group can comprise a cationic peptidyl group.In other embodiments, the cationic group can comprise a moiety (e.g., analkyl group or an alkylaryl group) substituted with a cationicsubstituent, such as an ammonium group (e.g., a tetraalkylammoniumgroup, a aryltrialkylammonium group, a diaryldialkylammonium group, or atriarylalkylammonium group) or a pyridinium group.

In some embodiments of Formula I and Formula IA, R² can comprise ahydrophobic group. For example, in certain embodiments, R² can comprisean aryl group or an alkylaryl group. In some embodiments, R² cancomprise a hydrophilic group. For example, in certain embodiments, R²can comprise a hydrophilic polyalkyleneoxy group (e.g., a polyethyleneoxide segment).

In some embodiments of Formula I and Formula IA, R² can comprise areactive functional group, such as an olefin, acetylene, alcohol,phenol, ether, oxide, halide, aldehyde, ketone, carboxylic acid, ester,amide, cyanate, isocyanate, thiocyanate, isothiocyanate, amine,hydrazine, hydrazone, hydrazide, diazo, diazonium, nitro, nitrile,mercaptan, sulfide, disulfide, sulfoxide, sulfone, sulfonic acid,sulfinic acid, acetal, ketal, anhydride, sulfate, sulfenic acidisonitrile, amidine, imide, imidate, nitrone, hydroxylamine, oxime,hydroxamic acid, thiohydroxamic acid, allene, ortho ester, sulfite,enamine, ynamine, urea, pseudourea, semicarbazide, carbodiimide,carbamate, imine, azide, azo group, azoxy group, nitroso group,N-hydroxysuccinimide ester, or maleimides. For example, in certainembodiments, R² can comprise an alkynyl group or an azido group. Inother embodiments, R² can comprise a hydrazone group.

In some embodiments of Formula I and Formula IA, n can be at least 2(e.g., at least 5, at least 10, at least 15, at least 20, at least 25,at least 30, at least 40, at least 50, at least 60, at least 70, atleast 80, at least 90, at least 100, at least 110, at least 120, atleast 130, at least 140, at least 150, at least 160, at least 170, atleast 180, at least 190, at least 200, at least 225, at least 250, atleast 275, at least 300, at least 325, at least 350, or at least 375).In some embodiments, n can be 400 or less (e.g., 375 or less, 350 orless, 325 or less, 300 or less, 275 or less, 250 or less, 225 or less,200 or less, 190 or less, 180 or less, 170 or less, 160 or less, 150 orless, 140 or less, 130 or less, 120 or less, 110 or less, 100 or less,90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less,30 or less, 25 or less, 20 or less, 15 or less, 10 or less, or 5 orless).

In can range from any of the minimum values described above to any ofthe maximum values described above. For example, n can range from 2 to400 (e.g., from 2 to 200, from 2 to 100, from 2 to 50, from 5 to 400,from 5 to 200, from 5 to 100, or from 5 to 50).

In other cases, the polycationic polymer can be formed by polymerizationof monomers (e.g., acrylate monomers) containing pendant cyclicbis-electrophiles (e.g., pendant versions of the cyclicbis-electrophiles defined by Formula IIA, Formula IIB, or Formula IICwhere Z is NR³, and R³ represents an acrylate group connected to thecyclic bis-electrophile via a linking group). Following polymerization,the pendant cyclic bis-electrophiles can be reacted with nucleophiles tointroduce positively charged centers within the polymer sidechains.

By way of example, also provided are polycationic polymers comprising arecurring unit defined by Formula V below

wherein R⁴ represents H or methyl; L is absent or represents a linkinggroup; A represents, individually for each occurrence, a heterocyclicring comprising a cationic nitrogen center; R⁵ is absent, or represents,individually for each occurrence, halogen, hydroxy, amino, cyano, azido,hydrazone, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, alkylaryl,alkylheteroaryl, cycloalkyl alkylcycloalkyl, heterocycloalkyl,alkylheterocycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, dialkylamino, alkylcarbonyl, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, peptidyl, polyamino, orpolyalkyleneoxy; and m is an integer from 0 to 5.

In some embodiments, L can be absent. In other embodiments, L is presentand represents a linking group. When present, the linking group can beany suitable group or moiety which is at minimum bivalent, and connectsthe pendant 9-azabicyclo[3.3.1]nonyl moiety to the polymer backbone. Thelinking group can be composed of any assembly of atoms, includingoligomeric and polymeric chains. In some cases, the total number ofatoms in the linking group can be from 3 to 200 atoms (e.g., from 3 to150 atoms, from 3 to 100 atoms, from 3 and 50 atoms, from 3 to 25 atoms,from 3 to 15 atoms, or from 3 to 10 atoms). In some embodiments, thelinking group can be, for example, an alkyl, alkoxy, alkylaryl,alkylheteroaryl, alkylcycloalkyl, alkylheterocycloalkyl, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, dialkylamino, alkylcarbonyl,alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, or polyaminogroup.

In Formula V, A can be any heterocyclic ring comprising a tertiarynitrogen atom. In some embodiments, A can be a heteroaromatic ring. Insome embodiments, A can comprise one or more additional heteroatoms inaddition to the tertiary nitrogen atom (e.g., one or more additionalnitrogen atoms, one or more oxygen atoms, one or more sulfur atoms, or acombination thereof). For example, A can be chosen from pyridine,imidazole, pyrazole, triazole, tetrazole, oxazole, isoxazole, furazan,isothioazole, and thiazole. In certain embodiments, A can represent apyridine ring.

In some embodiments, R⁵ can be absent. In other embodiments, R⁵ can bepresent. When present, R⁵ can comprise, individually for eachoccurrence, a halogen, hydroxy, amino, cyano, azido, hydrazone, alkyl,alkenyl, alkynyl, alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl,cycloalkyl alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, dialkylamino,alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,peptidyl, polyamino, or polyalkyleneoxy group.

In some embodiments, R⁵ can comprise a cationic group. In certainembodiments, the cationic group can comprise a cationic polyamino group(e.g., a polyethyleneimine segment). In other embodiments, the cationicgroup can comprise a cationic peptidyl group. In other embodiments, thecationic group can comprise a moiety (e.g., an alkyl group or analkylaryl group) substituted with a cationic substituent, such as anammonium group (e.g., a tetraalkylammonium group, a aryltrialkylammoniumgroup, a diaryldialkylammonium group, or a triarylalkylammonium group)or a pyridinium group.

In some embodiments, R⁵ can comprise a hydrophobic group. For example,in certain embodiments, R⁵ can comprise an aryl group or an alkylarylgroup. In some embodiments, R⁵ can comprise a hydrophilic group. Forexample, in certain embodiments, R⁵ can comprise a hydrophilicpolyalkyleneoxy group (e.g., a polyethylene oxide segment).

In some embodiments, R⁵ can comprise a reactive functional group, suchas an olefin, acetylene, alcohol, phenol, ether, oxide, halide,aldehyde, ketone, carboxylic acid, ester, amide, cyanate, isocyanate,thiocyanate, isothiocyanate, amine, hydrazine, hydrazone, hydrazide,diazo, diazonium, nitro, nitrile, mercaptan, sulfide, disulfide,sulfoxide, sulfone, sulfonic acid, sulfinic acid, acetal, ketal,anhydride, sulfate, sulfenic acid isonitrile, amidine, imide, imidate,nitrone, hydroxylamine, oxime, hydroxamic acid, thiohydroxamic acid,allene, ortho ester, sulfite, enamine, ynamine, urea, pseudourea,semicarbazide, carbodiimide, carbamate, imine, azide, azo group, azoxygroup, nitroso group, N-hydroxysuccinimide ester, or maleimides. Forexample, in certain embodiments, R⁵ can comprise an alkynyl group or anazido group. In other embodiments, R⁵ can comprise a hydrazone group.

In some embodiments, the polycationic polymer can further comprise arecurring unit derived from the polymerization of one or moreethylenically-unsaturated monomers. Example ethylenically-unsaturatedmonomers include (meth)acrylate monomers, vinyl aromatic monomers (e.g.,styrene), ethylenically unsaturated aliphatic monomers (e.g.,butadiene), vinyl ester monomers (e.g., vinyl acetate),(meth)acrylonitrile monomers, vinyl halide monomers, vinyl ethermonomers, silane-containing monomers, (meth)acrylamide monomers (as wellas (meth)acrylamide derivatives), sulfur-based monomers, andcombinations thereof.

Exemplary acrylate and (meth)acrylate monomers include, but are notlimited to, methyl acrylate, methyl (meth)acrylate, ethyl acrylate,ethyl (meth)acrylate, butyl acrylate, butyl (meth)acrylate, isobutyl(meth)acrylate, n-hexyl (meth)acrylate, ethylhexyl (meth)acrylate,n-heptyl (meth)acrylate, ethyl (meth)acrylate, 2-methylheptyl(meth)acrylate, octyl (meth)acrylate, isooctyl (meth)acrylate, n-nonyl(meth)acrylate, isononyl (meth)acrylate, n-decyl (meth)acrylate,isodecyl (meth)acrylate, dodecyl (meth)acrylate, lauryl (meth)acrylate,tridecyl (meth)acrylate, stearyl (meth)acrylate, glycidyl(meth)acrylate, alkyl crotonates, vinyl acetate, di-n-butyl maleate,di-octylmaleate, acetoacetoxyethyl (meth)acrylate, acetoacetoxypropyl(meth)acrylate, hydroxyethyl (meth)acrylate, allyl (meth)acrylate,tetrahydrofurfuryl (meth)acrylate, cyclohexyl (meth)acrylate,2-ethoxyethyl (meth)acrylate, 2-methoxy (meth)acrylate,2-(2-ethoxyethoxy)ethyl (meth)acrylate, 2-ethylhexyl (meth)acrylate,2-propylheptyl (meth)acrylate, 2-phenoxyethyl (meth)acrylate, isobornyl(meth)acrylate, caprolactone (meth)acrylate, polypropyleneglycolmono(meth)acrylate, polyethyleneglycol (meth)acrylate, benzyl(meth)acrylate, 2,3-di(acetoacetoxy)propyl (meth)acrylate, hydroxypropyl(meth)acrylate, methylpolyglycol (meth)acrylate,3,4-epoxycyclohexylmethyl (meth)acrylate, 1,6 hexanedioldi(meth)acrylate, 1,4 butanediol di(meth)acrylate and combinationsthereof.

Suitable vinyl aromatic compounds include styrene, α- andp-methylstyrene, α-butylstyrene, 4-n-butylstyrene, 4-n-decylstyrene,vinyltoluene, and combinations thereof. Vinyl esters of carboxylic acidsinclude, for example, vinyl laurate, vinyl stearate, vinyl propionate,versatic acid vinyl esters, vinyl acetate, and combinations thereof. Thevinyl halides can include ethylenically unsaturated compoundssubstituted by chlorine, fluorine or bromine, such as vinyl chloride andvinylidene chloride. The vinyl ethers can include, for example, vinylethers of alcohols comprising 1 to 4 carbon atoms, such as vinyl methylether or vinyl isobutyl ether. Aliphatic hydrocarbon monomers caninclude, for example, hydrocarbons having 4 to 8 carbon atoms and twoolefinic double bonds, such as butadiene, isoprene, and chloroprene.Silane containing monomers can include, for example, vinyl silanes, suchas vinyltrimethoxysilane, vinyltriethoxysilane (VTEO), vinyltris(2-methoxyethoxysilane), and vinyl triisopropoxysilane, and(meth)acrylatoalkoxysilanes, such as(meth)acryloyloxypropyltrimethoxysilane,γ-(meth)acryloxypropyltrimethoxysilane, andγ-(meth)acryloxypropyltriethoxysilane. (Meth)acrylamide derivativesinclude, for example, keto-containing amide functional monomers definedby the general structure belowCH₂═CR₁C(O)NR₂C(O)R₃wherein R₁ is hydrogen or methyl; R₂ is hydrogen, a C₁-C₄ alkyl group,or a phenyl group; and R₃ is hydrogen, a C₁-C₄ alkyl group, or a phenylgroup. For example, the (meth)acrylamide derivative can be diacetoneacrylamide (DAAM) or diacetone methacrylamide. Sulfur-containingmonomers include, for example, sulfonic acids and sulfonates, such asvinylsulfonic acid, 2-sulfoethyl methacrylate, sodium styrenesulfonate,2-sulfoxyethyl methacrylate, vinyl butylsulfonate, sulfones such asvinylsulfone, sulfoxides such as vinylsulfoxide, and sulfides such as1-(2-hydroxyethylthio) butadiene.

By way of non-limiting illustration, examples of certain embodiments ofthe present disclosure are given below.

EXAMPLES Example 1: Fragmentable Polycationic Materials Based onAnchimeric Assistance

New modular, fragmentable oligo- and polycations have been developedbased on the reactions of 9-thiabicyclo[3.3.1]dichloride and relatedcompounds with substituted dipyridyl nucleophiles by an anchimericassistance mechanism. Each bond-forming event in this condensationpolymerization process generates a positive charge in the main chain.Product lengths were found to be dependent on the reactivity of theelectrophile, which was tunable by changing the nature of the leavinggroup β to sulfur. The monomers were easily synthesized, and theresulting readily available polymers were found to be highly efficientbinders of nucleic acid. The polycations also exhibited properties ofcytotoxicity and DNA transfection with interesting structure-activitycharacteristics. The polycations decomposed by hydrolysis at ratesdependent on the leaving-group ability of the pyridyl unit, whichcorrelated roughly with the pK_(a) of its conjugate acid. Polymerdecomposition occurs simultaneously throughout the length of the chains,rather than from the ends; the decomposition products were tested andfound to be only minimally toxic to cultured cells

Background

The reliable substitution chemistry of 9-thiabicyclo[3.3.1]dichloride(1) and related electrophiles was first described almost 40 years ago bythe Weil, Corey, and Lautenschlaeger group (resulting in the designationof 1 as a “WCL” building block). The reactivity of this scaffold hasbeen explored for its fast and clean nature due to anchimeric assistanceprovided by the internal nucleophilic center. As with other reactionsmeeting the click chemistry standard, such reactions can potentially beused in materials science applications. Substitution chemistryaccelerated by neighboring group participation has been usedintermittently for many years in polymer synthesis and modification,although sometimes intramolecular effects have been mistaken for trueanchimeric assistance.

Cationic polymers are of interest primarily for gene delivery andsurface antimicrobial properties, with recent attention to new designsand precise control of structure-property relationships. The majority ofsuch materials employ primary, secondary, and tertiary amines thatrequire protonation to carry the desired charges.

Polyethylenimine(PEI), synthesized by the ring opening polymerization ofaziridine, is a popular example. Cleavable linkages such as esters,acetals/ketals, imines, and disulfides have been employed inconstructing biodegradable transfection agents from branched PEI, oftenwith the sacrifice of a unit positive charge at each point of linkerconnection. Permanently charged species such as quaternary ammonium orpyridinium ions are only rarely employed as linkages in the backbone ofpolycationic chains, although they have been used to modify polymerbranches and are the predominant positively-charged constituents ofcationic lipid head groups used as small-molecule transfection reagents.An example are the polyionenes usually synthesized by S_(N)2 Menschutkinreaction at comparatively high temperatures. The resulting materialsshow properties characteristic of other polycations, but are thermallyand chemically stable at physiological temperature, and are thereforenot biodegradable.

In this example, the use of WCL electrophiles in the synthesis of a newclass of polycationic molecules that have a unique architecture and modeof fragmentation are described. The polycationic materials arerepresented schematically in Scheme 1. In these systems, positive chargeis created by each substitution event and disappears when the linkagefragments in the course of anchimerically-assisted hydrolysis, ratherthan being carried by monomers, so the starting materials and productsare uncharged (except for byproduct mineral acid). Preliminarycharacterization of the cytotoxic and DNA transfection properties of thepolycations are also described, and demonstrate that this class ofmaterials may be suitable for antimicrobial and gene deliveryapplications.

Materials and Methods

Reagents and solvents were purchased from commercial sources and used asreceived, unless otherwise stated. When dry solvents were required,solvents were passed through activated alumina columns on an MBraunsolvent purification system (MB-SPS), and collected in oven-driedglassware prior to use. Water was purified on a Millipore Milli-QAdvantage A10 system. Unless otherwise stated, the reactions wereperformed under inert atmosphere in capped reaction vessels. Flashchromatography was performed on 60-mesh silica. Analytical TLC wasperformed on aluminum-backed plates and visualized by exposure to UVlight and/or staining with aqueous potassium permanganate (2% KMnO₄+5%K₂CO₃).

Instrumentation

NMR spectra were obtained on Bruker AMX-400, and DRX-500 instruments indeuterated solvents (Cambridge Isotope Laboratories, Inc.) andreferenced to the signals of residual protium in the NMR solvent.Spectra were processed in MestReNova software (Mestrelab Research).Routine mass spectra were obtained on an Advion Compact MassSpectrometer (G1946D) ESI-MSD instrument, using direct sample injectionfollowed with 9:1 CH₃CN:H₂O containing 0.1% formic acid as mobile phase.Absorbance and fluorescence spectra were collected on a VarioskanFlashplate reader (ThermoFisher). Gel permeation chromatography (GPC)analysis was performed in DMF or Milli-Q water at 1 mL/min flow rate(LC-20AD pump) on a Shimadzu GPC setup equipped with two PhenomenexPhenogel 10 μmlinear columns (300×7.8 mm) or PolySep-GFC-P (300×7.0 mm),autosampler (SIL-20A) and column oven (CTO-20A) set at 40° C. Detectionwas achieved using a diode array detector (SPD—M20A), and RI detector(RID—10A), and instrument was calibrated with polystyrene standards kits(Supelco) or Dextran kit (Phenomenex ALO-2772). Dynamic light scatteringmeasurements were taken on a DynaPro plate reader and analyzed withDynamics® software (Wyatt Technology, Santa Barbara, Calif.).Transmission electron microscope images were acquired on a HitachiHT7700 microscope operated at 120 kV.

Synthetic Procedures and Characterization of New Compounds

2,6-Dichloro-9-thiabicyclo[3.3.1]nonane (1a) was synthesized using themethods described in Diaz, D. D. et al.“2,6-Dichloro-9-thiabicyclo[3.3.1]nonane: Multigram Display of Azide andCyanide Components on a Versatile Scaffold,” Molecules, 2006, 11,212-218, which is hereby incorporated herein by reference. ¹H NMR (400MHz, CDCl₃) δ 4.71 (d, J=3.3 Hz, 2H), 2.85 (dd, J=6.8, 3.6 Hz, 2H),2.72-2.61 (m, 2H), 2.36-2.18 (m, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 62.34,37.10, 32.40, 28.12.

2,6-Dinitro-9-thiabicyclo[3.3.1]nonane (1b). 1a (21 mg, 0.1 mmol, 1equiv) and silver nitrate (34 mg, 0.2 mmol, 2 equiv) were mixed in 0.5mL MeCN and stirred overnight at room temperature. The resulting AgClwas removed by centrifugation and the solvent was evaporated to give 1bas a colorless oil (22 mg, 86% yield). ¹H NMR (400 MHz, CDCl₃) δ5.66-5.50 (m, 2H), 3.02 (d, J=3.1 Hz, 2H), 2.51 (dd, J=14.5, 6.7 Hz,2H), 2.32 (dd, J=9.8, 3.0 Hz, 2H), 2.20 (dt, J=13.5, 6.7 Hz, 2H),2.11-1.92 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 82.24, 32.72, 26.92,24.84.

2,6-Diiodo-9-thiabicyclo[3.3.1]nonane (1d). Compound 1a (252 mg, 1.2mmol, 1 equiv) and sodium iodide (540 mg, 3.6 mmol, 3 equiv) were mixedin 6 mL acetone and stirred overnight at room temperature. NaCl wasfiltered off and the solution was evaporated. The resulting solid wasdissolved in CH₂Cl₂, leaving behind excess sodium iodide. Evaporation ofthis solution gave 1d as a yellow powder (270 mg, 58% yield). ¹H NMR(500 MHz, CDCl₃) δ 5.26-5.12 (m, 2H), 3.11 (t, J=3.4 Hz, 2H), 2.95-2.81(m, 2H), 2.71 (ddd, J=11.6, 6.1, 4.0 Hz, 2H), 2.58-2.42 (m, 2H). ¹³C NMR(126 MHz, CDCl₃) δ 38.50, 36.11, 35.75, 32.35.

Representative procedure for the synthesis of pyridine adducts of bothsmall molecules and oligomers (Table 2, Table 3): Compound 1a (21 mg,0.1 mmol, 1 equiv), silver nitrate (34 mg, 0.2 mmol, 2 equiv) and thepyridine of interest (0.2 mmol, 2 equiv) were mixed in 0.5 mL MeCN andstirred overnight at room temperature under inert atmosphere. Theprecipitate was collected and washed with MeCN. The charged adduct wasdissolved in water and any remaining solids were removed bycentrifugation, repeating the centrifugation step until the solution wasnot cloudy. The resulting solution was frozen and lyophilized to obtainthe product as a colorless solid.

2,6-di-3-methoxycarbonylpyridinium-9-thiabicyclo[3.3.1]nonane dinitrate(5b): ¹H NMR (400 MHz, D₂O) δ 9.58 (s, 2H), 9.35 (d, J=4.9 Hz, 2H),9.23-9.08 (m, 2H), 8.47-8.27 (m, 2H), 5.85 (dd, J=8.3, 4.0 Hz, 2H), 3.41(s, 2H), 3.25 (q, J=13.0 Hz, 2H), 2.49 (t, J=14.5 Hz, 4H), 2.36 (d,J=14.9 Hz, 2H). ¹³C NMR (126 MHz, D₂O) δ 163.09, 146.47, 146.01, 144.54,131.16, 129.01, 74.35, 53.86, 35.67, 26.25, 24.27.

2,6-di-4-methoxycarbonylpyridinium-9-thiabicyclo[3.3.1]nonane dinitrate(5c): ¹H NMR (400 MHz, D₂O) δ 9.31 (d, J=6.7 Hz, 4H), 8.65 (d, J=6.6 Hz,4H), 5.92-5.77 (m, 2H), 3.40 (s, 2H), 3.19 (dd, J=12.9, 5.5 Hz, 2H),2.60-2.43 (m, 4H), 2.43-2.31 (m, 2H). ¹³C NMR (126 MHz, D₂O) δ 163.50,145.43, 144.84, 128.20, 74.39, 54.22, 35.92, 26.52, 24.68.

2,6-di-3-carbamylpyridinium-9-thiabicyclo[3.3.1]nonane dinitrate (5d):¹H NMR (400 MHz, D₂O) δ 9.47 (s, 2H), 9.28 (d, J=6.0 Hz, 2H), 8.98 (dd,J=16.1, 7.3 Hz, 2H), 8.48-8.28 (m, 2H), 5.94-5.74 (m, 2H), 3.41 (s, 2H),3.36-3.13 (m, 2H), 2.66-2.40 (m, 4H), 2.36 (d, J=15.7 Hz, 2H). ¹³C NMR(101 MHz, D₂O) δ 165.66, 145.33, 144.82, 143.44, 134.42, 128.93, 74.42,35.82, 26.42, 24.44.

2,6-di-4-carbamylpyridinium-9-thiabicyclo[3.3.1]nonane dinitrate (5e):¹H NMR (400 MHz, D₂O) δ 9.36 (d, J=6.8 Hz, 4H), 8.53 (d, J=6.7 Hz, 4H),5.87 (dd, J=10.5, 6.7 Hz, 2H), 3.45 (d, J=2.7 Hz, 2H), 3.36-3.17 (m,2H), 2.62-2.46 (m, 4H), 2.40 (dd, J=9.4, 6.4 Hz, 2H). ¹³C NMR (126 MHz,D₂O) δ 166.24, 149.13, 144.47, 126.79, 74.08, 35.78, 26.41, 24.59,

2,6-di-3-bromopyridinium-9-thiabicyclo[3.3.1]nonane dinitrate (5f): ¹HNMR (400 MHz, D₂O) δ 9.33 (d, J=0.9 Hz, 2H), 9.10 (d, J=5.2 Hz, 2H),8.93-8.72 (m, 2H), 8.11 (dd, J=8.3, 6.2 Hz, 2H), 5.90-5.61 (m, 2H), 3.34(s, 2H), 3.13 (dd, J=13.0, 5.7 Hz, 2H), 2.58-2.25 (m, 6H). ¹³C NMR (126MHz, D₂O) δ 149.30, 144.72, 141.88, 129.23, 123.63, 74.22, 35.83, 26.38,24.27.

2,6-di-3-trifluoromethylpyridinium-9-thiabicyclo[3.3.1]nonane dinitrate(5g): ¹H NMR (500 MHz, D₂O) δ 9.86 (s, 2H), 9.53 (d, J=6.4 Hz, 2H), 9.13(d, J=8.1 Hz, 2H), 8.53-8.48 (m, 2H), 6.01-5.89 (m, 2H), 3.52 (d, J=2.4Hz, 2H), 3.32 (dd, J=12.9, 6.2 Hz, 2H), 2.66-2.48 (m, 4H), 2.42 (dd,J=15.2, 4.3 Hz, 2H). ¹³C NMR (101 MHz, D2O) δ 149.76, 147.21, 147.17,129.58, 114.67, 113.24, 75.11, 35.64, 26.23, 24.36.

2,6-di-3-cyanopyridinium-9-thiabicyclo[3.3.1]nonane dinitrate (5h): ¹HNMR (500 MHz, D₂O) δ 9.69 (s, 2H), 9.54 (d, J=6.3 Hz, 2H), 9.07 (d,J=8.2 Hz, 2H), 8.61-8.44 (m, 2H), 5.94 (dd, J=8.3, 3.7 Hz, 2H), 3.50 (d,J=3.0 Hz, 2H), 3.37 (dd, J=12.9, 6.4 Hz, 2H), 2.66-2.45 (m, 4H),2.45-2.33 (m, 2H). ¹³C NMR (101 MHz, D₂O) δ 146.26 (s), 144.09 (d, J=3.0Hz), 142.46 (d, J=4.0 Hz), 131.43 (q, J=36.5 Hz), 121.26 (q, J=273.2Hz), 75.01 (s), 35.82 (s), 26.42 (s), 24.34 (s).

2,6-di-3-bromo-5-methoxylpyridinium-9-thiabicyclo[3.3.1]nonane dinitrate(5i): ¹H NMR (400 MHz, D₂O) δ 9.00 (s, 2H), 8.87 (s, 2H), 8.49 (s, 2H),5.79-5.63 (m, 2H), 3.41 (d, J=2.6 Hz, 2H), 3.27 (dd, J=13.0, 6.1 Hz,2H), 2.49 (td, J=11.3, 6.4 Hz, 2H), 2.45-2.31 (m, 4H). ¹³C NMR (126 MHz,D₂O) δ 159.03, 135.88, 133.04, 131.50, 123.55, 74.47, 57.72, 35.92,26.48, 24.03.

2,6-di-3- bromo-5-carbamylpyridinium-9-thiabicyclo[3.3.1]nonaneditriflate (5j): ¹H NMR (500 MHz, D₂O) δ 9.47 (s, 2H), 9.41 (s, 2H),9.17 (s, 2H), 5.77 (dd, J=8.4, 4.0 Hz, 2H), 3.37 (s, 2H), 3.19 (dd,J=13.1, 6.1 Hz, 2H), 2.46 (t, J=11.3 Hz, 2H), 2.38 (dd, J=13.2, 6.1 Hz,2H), 2.34-2.25 (m, 2H). ¹³C NMR (126 MHz, D₂O) δ 164.26, 147.54, 146.69,141.73, 134.73, 123.69, 74.79, 35.62, 26.07, 23.87.

2,6-di-3,5-dimethoxycarbonylpyridinium-9-thiabicyclo[3.3.1]nonaneditriflate (5k): ¹H NMR (400 MHz, D₂O) δ 9.70 (s, 4H), 9.50 (s, 2H),5.89 (d, J=12.5 Hz, 2H), 3.37 (s, 2H), 3.30 (dd, J=12.7, 5.2 Hz, 2H),2.53-2.21 (m, 6H).

2,6-di-3-bromo-5-methoxycarbonylpyridinium-9-thiabicyclo[3.3.1]nonaneditriflate (5l): ¹H NMR (400 MHz, D₂O) δ 9.51 (s, 2H), 9.48 (s, 2H),9.28 (s, 2H), 5.75 (d, J=12.1 Hz, 2H), 3.32 (s, 2H), 3.17 (ddd, J=25.6,13.2, 6.3 Hz, 2H), 2.51-2.18 (m, 6H).

Synthesis of bispyridine linker 2i: 5-Bromopyridin-3-ol (570 mg, 3.3mmol, 4 equiv) was dissolved in 5 mL of 1 M NaOH with vigorous stirringfor 1 h. Propane-1,3-diyl bis(4-methylbenzenesulfonate) (320 mg, 0.83mmol, 1 equiv) and tetra-n-butylammonium bromide (367 mg 1.14 mmol, 1.3equiv) in 10 mL toluene were added. The solution was heated to 95° C.overnight. The mixture was cooled, the solvent removed by rotaryevaporation, and the product was purified by column chromatography (2:1hexanes/EtOAc) elution. The desired product,1,3-bis((5-bromopyridin-3-yl)oxy)propane (2i), was obtained as a whitepowder (216 mg, 67% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.32 (d, J=1.6 Hz,1H), 8.27 (d, J=2.5 Hz, 1H), 7.41 (s, 1H), 4.23 (t, J=5.9 Hz, 2H),2.39-2.29 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 154.99, 143.04, 136.22,123.70, 120.26, 64.48, 28.71.

Representative procedure for the synthesis of polymers (entries 4-10 inTable 4 and Table 5). Compound 1a (21 mg, 0.1 mmol, 1 equiv), silversalt (0.2 mmol, 2 equiv) and the bispyridine of interest (0.1 mmol, 1equiv) were mixed in 0.5 ml MeCN and stirred overnight at roomtemperature under inert atmosphere. For aqueous soluble polymers(entries 3, 4, 5, and 7 in Table 4; entries 1-3 in Table 5), theprecipitate was collected and washed with MeCN. The charged adduct wasdissolved in water and any remaining solids were removed bycentrifugation, repeating the centrifugation step until the solution wasnot cloudy. The resulting solution was frozen and lyophilized to obtainthe product as a colorless solid. For organic soluble polymers (entries6, 8, 9, and 10 in Table 4; entry 4 in Table 5), any solid material wasfiltered out and the resulting solution was added to 10 ml CH₂Cl₂ toprecipitate the desired product. The material was isolated by filtrationand dried to give an off-white solid.

-   -   Alternative Strategies for Accelerating the Substitution        Chemistry of Compound I

Different halophilic transition metals were tested for their ability tospeed the substitution reactions of 1 by interaction with the leavinggroup, accelerating the rate-limiting formation of the strainedepisulfonium intermediate. Representative Al, Sn, and Pd species showedno effect (Table 1). Silver salts were quite effective, prompting theisolation of the resulting intermediates as described below.

TABLE 1 Survey of halophilic metal complexes for the acceleration ofreactions of 1 with PhCH₂NH₂ (10 equiv) in acetonitrile at roomtemperature. Reaction Entry Halophilic Metal Complex Time 1 None 16 h 2AgOTf 2 h 3 AgNO₃ 4 h 4 AlCl₃ >16 h 5 SnCl₂ 14 h 6 PdCl₂ 12 h 7 Pd(OAc)₂14 h 8 Zr(Cp)₂Cl₂ >16 h 9 Pd(PPh₃)₂Cl₂ >16 h

Comparison of Reactivities Between Different WCL Intermediates

Compound 1a (10.5 mg, 0.05 mmol, 1 equiv) and 1b (0.2 mL of 0.25 M MeCNsolution, 0.05 mmol, 1 equiv) were combined in 1 mL MeCN. The solventwas then evaporated and the residue analyzed by ¹H NMR in CDCl₃ (FIG. 2, spectra A). In parallel, a separate identical [1a+1b] mixture in MeCNwas treated with benzylamine (8.1 μL, 0.1 mmol, 1 equiv) andtriethylamine (69.6 μL, 0.5 mmol, 5 equiv, previously shown to beunreactive towards WCL compounds and used to soak up any HCl produced).After 24 hours, the mixture was evaporated, redissolved in CDCl₃, andanalyzed by ¹H NMR (FIG. 2 , spectra B). The data show almost completereaction of the amine with the nitrate electrophile rather than thechloride.

An internal competition between all three easy-to-handle electrophiles(chloride, nitrate, iodide; 1a, 1b, and 1d) was performed in a similarmanner, in which 0.05 mmol of each was combined in acetonitrile andtreated with slightly less than one equivalent of BnNH₂ (0.125 mmol) and5 equiv. (5 mmol) of Et₃N (total volume 1 mL). NMR spectra recordedimmediately and after 24 h (in each case, an aliquot was removed,evaporated, and redissolved in CDCl₃) are shown in FIG. 3 . Integrationof the α-protons for each electrophile showed nearly equimolar ratios atthe start, and a highly skewed distribution after reaction. The relativereactivities thus revealed are iodide>nitrate>chloride.

Monitoring Fragmentation of Representative WCL-Substituted Adducts andWCL-Based Oligocations by ¹H NMR

The fragmentation of cationic adducts was followed by ¹H NMR indeuterated buffer, with the relative concentration of each speciesestablished by integration of unique resonances. Each adduct in Table 3and Table 4 was thus examined in three independent runs.

FIGS. 4A-4B show representative fragmentation data for compound 5d, withthe relative concentration of each species established by integration ofunique resonances, labeled in the ¹H NMR spectra. The first-orderkinetic treatment is shown in FIG. 5 .

FIG. 6 shows representative fragmentation data for polymer 3i, with therelative concentration of each species established by integration ofunique resonances, labeled in the ¹H NMR spectra.

GPC Analysis of WCL Polymers Before and After Fragmentation

Three WCL based polymers were dissolved in pure water at 1 mg/mLconcentration solution and characterized by aqueous gel permeationchromatography (GPC, black traces in FIGS. 7A-7C). Separate identicalsolutions were heated at 37° C. for 36 hours and analyzed again byaqueous GPC (gray traces in FIGS. 7A-7C). The major peaks between 8 and10 mins were assigned to polymers; after heating, small moleculecomponents were evident as peaks at longer retention times. Polymer 3awas confirmed as being a particularly stable molecule, showing nodifference in GPC trace after heating. Polymers 3c and 3i underwentpartial degradation as expected from NMR experiments; interestingly, theremaining polymer peaks shifted modestly but reproducibly to shorterretention time (higher molecular weight, FIGS. 7B-7C). This couldsuggest (a) some ligation of oligomers (bearing pyridine nucleophiles atthe end) with active episulufonium electrophilic intermediates formed inthe decomposition process, and/or (b) the presence of cyclic structuresof sufficient size to result in a shift to higher molecular weight uponpartial cleavage. Calculation of molecular weight parameters bycomparison to dextran standards proved to be nonsensical, so Mn, Mw, andpolydispersities could not be assigned by this method.

Thermal Analysis of Representative WCL Polymers

The thermal decomposition temperature of WCL polymers were measured on aPerkin-Elmer Pyris 1 thermogravimetric analyzer (TGA) in a nitrogenatmosphere (flow rate 25 mL/min) with a heating rate of 10° C./min.Thermal transitions of WCL polymer 3a were measured with a TA Q200Differential Scanning calorimeter (DSC) in a nitrogen atmosphere (flowrate 50 mL/min) with a heating/cooling rate of 10° C./min Each samplewas scanned for three cycles and the 2^(nd) cycle is presented in FIG.8B.

Polymer 3a began extensive decomposition at approximately 220° C. whilethe decomposition of 3c and 3i was initiated at approximately 180-185°C., with two obvious stages of mass loss. A glass transition of 3a wasobserved at 67° C., comparable to Tg values previously reported forrepresentative polyionenes.

Cell Culture

All cell culture reagents were acquired from Life Technologies(Carlsbad, Calif.) unless noted otherwise. HeLa cells were purchasedfrom ATCC (Manassas, Va.). GFP-HeLa cells were a gift from the Schmidlab (University of Texas, Southwestern, Dallas, Tex.). Cells were grownand maintained in complete growth media: Dulbecco's Modified Eagle'sMedium (DMEM), supplemented with 5% Fetal bovine serum (FBS), sodiumpyruvate (1 mM), penicillin (100 units/mL), streptomycin (100 μg/mL),and GlutaMAX (2 mM). Cells were grown at 37° C. under humidified airwith 5% CO₂. Glass-bottomed culture dishes were purchased form MatTek(Ashland, Mass.). Accutase was purchased from Innovative CellTechnologies, Inc. (San Diego, Calif.). 96-well microtiter plates werepurchased from Thermo Fisher (Waltham, Mass.).

siRNAs were a gift from Integrated DNA Technologies (Coralville, Iowa),with the following sequences. GFP sense: 5′-CAAGCUGACCCUGAAGUUCUU (SEQID NO: 1), GFP antisense: 5′-GAACUUCAGGGUCAGCUUGUU (SEQ ID NO: 2).Approximately 1×10⁴ GFP-HeLa cells/well were plated in a 96-well platein 100 μL of complete growth media, and adhered overnight at 37° C. Forpositive control experiments, 10 nM double-stranded siRNA wastransfected with Lipofectamine RNAiMAX according to the manufacturer'sprotocol. After 24 h, the media was replaced with complete growth media(100 μL/well). Fluorescence was measured 48 h post-transfection. Forknockdown experiments with polyplexes, cells were plated as described.100 μL of oligomer/ds-siRNA complexes at indicated N/P ratios were addedto cells. Media was again replenished after 24 h. Fluorescence wasmeasured 48 h post-treatment.

Viability of Mammalian Cells in the Presence of WCL Monomers andPolymers

Approximately 1×10⁴ CHO-K1 cells/well were plated in a 96-well plate in100 μL of complete growth media, and adhered overnight at 37° C. Themedia was then replaced by 100 μL of polymer or monomer in serum-freemedia at the indicated concentration. Full media was replenished after 4or 24 h. Viability was measured by MTT assay, with non-treated cellsassigned as 100% viability. The results are plotted in FIGS. 11A-11D and12A-12H.

Results and Discussion

Fundamental Reactivity

While the substitution chemistry of compound 1a (Scheme 1, X=Cl) isquite clean, its reaction rate at room temperature (rate constant2.4×10⁻⁴ M⁻¹s⁻¹) and thermodynamic driving force were thought to beincompatible with the generation of high molecular weight polymers bythe simple condensation route outlined in Scheme 1. Accordingly, morereactive forms of 1 were prepared, focusing on the rate-limitingformation of episulfonium ion intermediate. Treatment of 1a with silvernitrate or silver triflate in acetonitrile afforded 1b or 1c in highyield after filtration of AgCl. The triflate 1c proved to be the mostreactive, but may have limited utility under practical conditionsbecause of its high sensitivity to hydrolysis with atmospheric moistureor trace water in the solvent. The nitrate derivative 1b, in contrast,was stable enough to survive standard isolation, handling, NMRcharacterization. The apparent Finkelstein reaction of 1a with sodiumiodide in acetone gave 1d in high yield, which was similarly stable toisolation by filtration and characterization in air. The NMR spectrarevealed significant differences between the chemical shifts of the Hatoms adjacent to the leaving groups (on C1 and C5) among the differentderivatives, suggesting that the order of reactivity would be iodide(1d)≥nitrate (1b)>chloride (1a), correcting for the upfield shiftcharacteristic of C—H bonds next to iodine due to factors unrelated tothe electron density of the C-halogen bond.

A preliminary assessment of substitution reactivity was obtained bycompetition reactions in which an equimolar mixture of two electrophileswas treated with half an equivalent of benzylamine (FIG. 1 ). Thesepairwise comparisons, as well as reaction of all three electrophilestogether (FIG. 2 , FIG. 3 ) confirmed the above order of reactivity,with nitrate approximately 10 times more reactive than chloride, andiodide at least 5 times more reactive than nitrate. These differencesare expected to be general since the reaction rate is only weaklydependent on the nature of the capturing nucleophile.

The parent pyridine derivative 5a was highly stable toward fragmentationin water (less than 5% hydrolysis observed after 1 week at 50° C.), sopyridine nucleophiles were chosen to include primarilyelectron-withdrawing groups to enhance this rate. Such pyridinederivatives are necessarily poorer nucleophiles for the forward step, sothe enhanced reactivity of 1b was important for the efficient synthesisof the corresponding substituted pyridine dicationic adducts, as shownin Table 2. Each adduct dinitrate was isolated, characterized, andheated at 50° C. in deuterated buffer; following the disappearance ofthe adduct by NMR, first-order rate constants of fragmentation wereobtained. The NMR spectra also confirmed the clean nature of thereaction, giving diol 4 and the released pyridines.

TABLE 2 Comparison of pseudo first order fragmentation of pyridineadducts at 50° C.; pK_(a) refers to the conjugate acid of the pyridinecalculated using Advanced Chemistry Development (ACD/Labs) SoftwareV11.02 ( © 1994-2015 ACD/Labs).

Compound R pK_(a) k_(frag) (h⁻¹) k_(rel) half life (h) 5a H 5.23      <1× 10⁻⁴ — — 5b 3-CO₂Me 3.19 (3.03 ± 0.38) × 10⁻² 1.86 23.2 ± 3.1 5c4-CO₂Me 3.16 (3.66 ± 0.33) × 10⁻² 2.25 19.1 ± 1.8 5d 3-CO₂NH₂ 3.54 (3.18± 0.32) ×10⁻² 1.95 21.7 ± 1.8 5e 4-CO₂NH₂ 3.39 (1.62 ± 0.10) ×10⁻² 1.0042.7 ± 2.7 5f 3-Br 2.87 (6.59 ± 0.58) × 10⁻² 4.05  9.9 ± 1.1 5g 3-CN1.78  (74.8 ± 5.4) × 10⁻² 46.2  0.9 ± 0.1 5h 3-CF₃ 2.8 (10.8 ± 0.98) ×10⁻² 6.68  6.4 ± 0.8 5i 3-Br, 5-OMe 2.26 (7.51 ± 0.53) × 10⁻² 4.64  9.2± 0.8

The stability of pyridine adducts to fragmentation in water was found toroughly correlate with calculated pyridine basicity (FIGS. 13A-13B).This is consistent with the idea that more nucleophilic pyridine groupsare more difficult to eject from the pyridinium adduct structure, andthat Brønsted basicity is indicative of nucleophilicity. In practice,half-lives were shown to vary over a 40-fold range (Table 2).Decomposition rates at 37° C., tested for potential applications invivo, were found to be much slower than at 50° C., as expected for afragmentation reaction. For example, 5c underwent hydrolysisapproximately 23 times faster at 50° C. than at 37° C. To tune thesepotential release rates to a reasonably fast range, twoelectron-withdrawing groups were used, leaving the ortho positionsunsubstituted to avoid steric problems in adduct formation (Table 3).Some additional limits were imposed on the process by the observationthat pyridines having too many (or too potent) electron-withdrawingsubstituents were found not to react cleanly with 1b or 1d. For example,the strongly electron-deficient 3-bromo-5-amidopyridine (calculatedpK_(a)=1.22) did not form a clean adduct unless reacted with the mostreactive ditriflate electrophile, 1c. The resulting compound 5junderwent relatively fast fragmentation (half life less than a day), asexpected. Carboxylate-based electron-withdrawing groups provide for easylinkage of pyridine units together to make higher-valent monomers forthe synthesis of fragmentable polymers and crosslinked materials.

TABLE 3 Comparison of pseudo first order fragmentation of pyridineadducts at 37° C. Com- pound R pK_(a) k_(frag) (h⁻¹) k_(rel) half life(h) 5c 4-CO₂Me 3.16 (0.16 ± 0.01) × 10⁻² 0.3  422 ± 41 5g 3-CN 1.78(5.87 ± 0.71) × 10⁻² 11 11.8 ± 1.9 5h 3-CF₃ 2.8 (1.14 ± 0.16) × 10⁻² 2.1  61 ± 8 5i 3-Br, 2.26 (0.55 ± 0.04) × 10⁻² 1  127 ± 10 5-OMe 5j 3-Br,1.22 (9.34 ± 0.87) × 10⁻² 17  7.4 ± 0.5 5-CO₂NH₂ 5k 3-Br, 0.84 (11.9 ±1.13) × 10⁻² 22 0.24 ± 0.01 5-CO₂Me 5l 3-CO₂Me, 1.16 (10.9 ± 1.81) ×10⁻² 20 0.27 ± 0.01 5-CO₂Me

Oligo- and Polycation Synthesis

The condensation polymerization of a dipyridine nucleophile and WCLelectrophile was explored using the parent compound 2a (Scheme 1, Table4). Reaction of 1a and 2a in MeCN solvent at 50° C., conditions thatproduce good yields of adducts with pyridine itself, give only smalloligomers (average n=3). In contrast, dinitrate 1b, generated in situfrom 1a and AgNO₃, gave copious quantities of precipitate within twominutes of mixing with an equimolar amount of 2a, all in MeCN.Filtration and washing provided the corresponding species 3a·(NO₃)_(2n).Characterization of this material was aided by its high solubility inwater and by the fact that the second substitution event on each WCLcore is usually faster than the first, so that chains are capped by thepyridine nucleophile. ¹H NMR in D₂O therefore showed distinct residuesfor non-alkylated pyridines at each end, enabling an estimate of averagechain length of approximately n=10 for 3a prepared under theseconditions (molecular weight approximately 4000). Gel permeationchromatography (GPC) analysis also showed minor fractions with longerretention time, consistent with shorter chains or cyclic oligomers.Somewhat higher molecular weights were obtained with heating to 50° C.(n≈13), or with the use solvents better able to support the developingpolycation and therefore to delay precipitation until longer chainlengths are achieved (Table 3). In a brief survey of activating silversalts and solvents, the use of AgPF₆ in DMSO was found to give thelargest average molecular weights as determined by NMR end-groupanalysis (Table 4); polydispersities could not be assessed because ofthe lack of suitable GPC standards (values obtained against dextranstandards in water were wildly divergent from estimates made by NMR) andbecause m/z values for molecules of different chain lengths are closeenough to make deconvolution of the mass spectra of these mixturesdifficult. Values of hydrodynamic radii determined by dynamic lightscattering (Table 6) were comparable with those of ammonium ionenes withsimilar degrees of polymerization. Analysis by TGA showed representativeoligomers to have the anticipated stability (decomposition initiation atapproximately 200° C.), and DSC revealed discrete glass transitions tobe rare, both consistent with materials that are potentially dynamic athigher temperatures. Note that the WCL electrophiles are used here inracemic form, making for polymeric structures that are likely mixturesof many diastereomers.

TABLE 4 Condensation polymerization of 1a + 2a. [1a] = [2a] AdditiveM_(n) Entry (M) (2 equiv) Solvent Temperature (kDa) n^(a) 1 0.1 NoneMeCN 50° C. 1.5  3 2 0.1 None THF/H₂O RT — — 3 0.1 AgNO₃ MeCN RT 4-58-10 4 0.1 AgNO₃ MeCN 50° C. 6 13 5 0.1 AgNO₃ THF/H₂O RT — — 6 0.1 AgBF₄DMF RT 5.3 10 7 0.2 AgNO₃ DMSO RT 9 18 8 0.2 AgOTf DMSO RT 11.5 23 9 0.4AgPF₆ DMSO RT 22.2 35 10 0.2 AgBF₄ DMSO RT 11.4 22 ^(a)n = averagedegree of polymerization

Using the conditions of Table 4, three additional oligocations wereprepared from the bis(pyridine) linkers as shown in Table 5. Thesynthetic procedure proved to be modular, giving oligomers of similarsize (n≈10) with the same methods of isolation and analysis. Thestabilities of these compounds toward fragmentation under aqueousconditions were assessed by NMR in deuterated buffer, following thedisappearance of the thiabicyclononane C—H resonance adjacent to thepyridine group (position g in FIG. 14 ) and the appearance of thecorresponding resonance (4.3 ppm) for the diol product 4 (FIG. 6 ). Theobserved half lives of the WCL-pyridine bonds in these oligomerscorresponded reasonably well (within a factor of 2) with the analogoussmall-molecule bis(pyridine) adducts described above, ranging fromseveral hours to a month at physiological temperature and pH. Note ofcourse that the oligomer structures decompose much faster than this,since the rupture of only a fraction of the bonds (occurring atapproximately the same first-order rate at each position of the chain)would be necessary to chop the linear structure into shorter pieces.Indeed, these materials are also very likely to be dynamic in nature,since breakage of a C—N(pyridyl) bond is probably followed byre-formation of that same bond more often than by irreversible captureof the intermediate episulfonium ion with water.

TABLE 5 Representative oligocations and half lives (t_(1/2)) of C—N bondfragmentation in aqueous buffer (pH 7). Nucleophilic Monomer t_(1/2) ofSmall [Polymer] n ^(a) T ^(b) t_(1/2) Molecule Analogue

10 37° C. 50° C. n.d. n.d. n.d. n.d.

10 37° C. 50° C. 624 h 36 h 422 h (5c) 19 h (5c)

6 37° C. 70 h 127 h at 37° C. (5i)

6 37° C. 9.2 h 5.8 h (5j) (a) n = average degree of polymerization. (b)Temperature at which polymer decomposition was measured in aqueousbuffer. Polymer nitrates (3a, 3c, 3i) were prepared as in Table 4, entry3; the triflate 3j was prepared as in Table 4, entry 8. ″n.d.″ = nodecomposition observed by NMR (<5%) after 1 week at the indicatedtemperature.

TABLE 6 Characterization of polymer/polyplexes formed from the indicatedpolycations (1 mg/ml in PBS) or polycations and double-stranded plasmidDNA (pcDNA3-EGFP, 6.2 kb, 20 ng/μL) at the indicated N/P ratio. Radiusand polydispersity were determined by dynamic light scattering. Cationicradius pd Entry Oligomer N/P Conditions (nm) (%)  1 3a•(NO₃)_(n)  2 H₂O,RT, freshly  82 ± 2 19.7 prepared  2 3a•(NO₃)_(n)  2 Entry 1, heated at37° C.  80 ± 3 16.5 for 48 h  3 3a•(NO₃)_(n)  2 PBS buffer, RT, freshly147 ± 9 15.5 prepared  4 3i•(NO₃)_(n)  2 H₂O, RT, freshly 116 ± 6 30.5prepared  5 3i•(NO₃)_(n)  2 Entry 4, heated at 37° C. n/o n/o for 48 h 6 3i•(NO₃)_(n)  2 PBS buffer, RT, freshly 210 ± 18 37.8 prepared  75a•(NO₃)₂ 150 H₂O or PBS n/o n/o  8 none — Plasmid DNA alone n/o n/o  93a•(NO₃)_(n) — PBS buffer, RT, no DNA  31 ± 2 17.6 10 3c•(NO₃)_(n) — PBSbuffer, RT, no DNA  56 ± 4 21.4 11 3i•(NO₃)_(n) — PBS buffer, RT, no DNA 49 ± 3 12.4 12 3i•(NO₃)_(n) — Entry 11, heated at 50° C. n/o n/o for 4h “pd” = polydispersity; “n/o” = no aggregates were observed.

-   -   Binding and Transfection of DNA and siRNA

Binding of WCL oligocations to DNA was examined by agarose gel shiftassay; a representative example is shown in FIG. 15A. Polyplex formationat a level sufficient to completely inhibit the electrophoreticmigration of a 6.2-kb double-stranded plasmid was observed with polymer3a at a low N/P ratio of approximately 1.0. A similar result wasobserved for 3i, but polymer 3c was less efficient, showing significantbut incomplete retardation of electrophoresis at N/P=50. Dynamic lightscattering analysis showed the resulting polyplexes to be of reasonablesize and narrow size distribution (Table 6), with larger structuresformed in PBS buffer vs. water. The presence of small-molecule adduct 5ain large excess had no effect on the electrophoresis of DNA nor was anobservable polyplex formed. The polyplex formed with 3a resisted heatingat 37° C. for 48 hours, whereas particles formed with 3i were decomposedafter 24 hours of such treatment (Table 6, entries 2,5), correspondingto the fragmentation stabilities observed for the polycations alone(Table 5).

The efficiency of both stable and fragmentable oligomers for thetransfection of siRNA into HeLa cells was surveyed as shown in FIGS.16A-16B. Much higher N/P ratios were required for siRNA transfectionthan for DNA complexation, and were constrained by the cytotoxicity ofthese new materials.

As shown in FIGS. 11A-11C, polymers 3a, 3c, and 3i engenderedsignificant toxicity after 24 hours exposure at 2 μM, 200 μM and 12 μMrespectively (concentrations refer to that of the chains, not themonomeric units, calculated based on average molecular weightsestablished by NMR); toxicities were much lower for 4 h exposure. Themilder toxicity of 3c could be due to ester cleavage in the cell orculture media. Polymers 3a and 3c should not fragment significantly byanchimeric substitution during 24 h incubation at 37° C.; 3i mayexperience significant fragmentation over that time, since C—N bondbreakage should occur to approximately 21% after 24 h. In any event, thedipyridine monomers from which these polymers are constructed (2a, 2c,and 2i) were found to be nontoxic at concentrations below 500 μM, andthe other decomposition product (diol 4) had no effect on cellularviability up to 1 mM. Interestingly, the dicationic monomeric adducts5a, 5c, and 5i were much less toxic than their corresponding polymers(no effect up to 500 μM or greater), and a longer polymer (3a′,approximately 18 repeat units; Table 4, entry 7) was significantly moretoxic than 3a (approximately 10 repeat units) at 4 h exposure. Theseobservations suggest that cytotoxicity is associated with the polymericnature of these materials and therefore results from interactions withthe cell membrane, rather than being a function of DNA alkylation.

At optimal N/P ratios of 55-65, polymer 3a was able to mediatetransfection with efficiencies near that of a standard commercialreagent (Lipofectamine, RNAiMAX) with similarly modest cytotoxicity(FIG. 16A), but these materials are, as discussed above, more toxicoverall toward cultured cells. Polymer 3i exhibited no separationbetween cellular toxicity and apparent siRNA knockdown (FIG. 16B).Preliminary show that transfection and expression of plasmid DNA in HeLacells can also be mediated by polymer 3a.

Conclusions

A new class of fragmentable polycations has been developed throughfacile nucleophilic substitution of WCL scaffolds by divalent pyridines.The efficiency of this process depends on the leaving group ability ofthe group being substituted, consistent with rate-limiting activation ofthe electrophile by formation of a highly reactive episulfonium ion.Degrees of polymerization from approximately 3 to 30 were observed forthese condensation polymerization processes. A range of polymerstabilities could be engineered by tuning the nucleophilicity andleaving group ability of the pyridine units, with pyridine basicityserving as a reasonably good guide to these properties. The resultingpolymers were highly efficient in complexing DNA and showed functionalbehavior (transfection and cytotoxicity) associated with some otherclasses of polycationic materials.

Example 2: Preparation of Polycations Bearing Cationic Side ChainsFormed by the Condensation of 9-azabicyclo[ 3.3.1]nonyl Electrophileswith Nucleophiles

Polycationic polymers bearing cationic side chains formed by thecondensation of a 9-azabicyclo[3.3.1]nonyl electrophile withnucleophiles were prepared using the synthetic methodology outlined inSchemes 2 and 3.

Using the synthetic methodology detailed in schemes 2 and 3, thepolymers below were successfully prepared.

Example 3: Cytotoxicity Activity of Polycationic Materials

As detailed in Example 1, condensation of9-thia/aza/selenabicyclo[3.3.1]nonyl electrophiles withpolynucleophiles, with each bond-forming event creating a positivelycharged center, provides access to a wide range of polycationicmaterials. These materials are subject to fragmentation by the reverseprocess, in which the internal nucleophile ejects a heterocyclicnucleophile as a leaving group. In the case of pyridines, fragmentationrates can be efficiently tuned from hours to years at 37° C.

The cytotoxic function of these polycations was evaluated by twomethods:

Method A

Bacteria suspensions (E. coli and B. subtilis ATCC 8037) were grown inMueller-Hinton Broth (MHB) overnight at 37° C. The resulting culture wasused to inoculate a second culture in 2 mL of MHB medium until anoptical density of 0.8 at 600 nm was reached. The suspension was dilutedwith fresh MHB to an optical density of 0.001 (approximately) at 600 nm(OD600). This suspension was mixed with different concentrations offreshly prepared polymer solutions in TRIS saline (pH 7.0) in a 96-wellplate, and incubated overnight at 37° C. The OD₆₀₀ was measured forbacteria suspensions that were incubated in the presence of polymersolution or only buffer solutions as controls. Antibacterial activitywas expressed as minimal inhibitory concentration (MIC), theconcentration at which more than 90% inhibition of growth was observed.All experiments were run in triplicate.

Method B

The bacteria were collected by centrifugation at 4,000×g for 3 min at 4°C., washed with sterile PBS (pH 7.4) and finally suspended in PBS to geta final concentration of 6×10⁶ cells mL⁻¹. The polymer solution wasadded into the bacteria suspension and shaken for 0.5-4 hours. Then 25μL of suspension was spread onto a sterile Petri dishes covered with alayer of LB medium containing 0.8% agar (previously autoclaved, andcooled to 37° C.). After overnight incubation at 37° C., bacterialcolonies became visible and were counted and compared with the untreatedbacteria plate. The MIC was defined as the minimum concentration in thediluted series when the CFU number on the agar plate reached no morethan 10% of the control plate.

As shown in FIG. 16 , the polycations exhibited extraordinary cytotoxicactivity, with E. coli MIC values for the polymers in solution at orbelow 0.1 μg/mL. These polycations are more than two orders of magnitudemore potent than standard antibacterial polymers or compounds.Measurements conducted against Gram-positive B. subtilis gave similarresults. Polycations employing the same type of positive charge carrierarranged on the side chains of acrylate monomers (as opposed to withinthe polymer backbone) were much less active, even though the chargedensity per molecular weight is nearly identical. Furthermore, thepolycations were less toxic toward a representative mammalian cell lineby 100-fold or more.

Members of this class are also efficient at binding polynucleotides,forming discrete polyplexes at 1:1 positive:negative charge ratios,which are far lower than most polycationic transfection agents.Monomeric adducts analogous to some of the active polymers were alsotoxic, but significantly less so.

Example 4: Covalent Attachment of Polycations to Glass Substrates

Remarkable levels of bacterial cytotoxicity were also observed when thepolycations described above were covalently attached to the surface ofsubstrates. In this example, the polycations were covalently attached toa glass substrate. The strategy used to immobilize the polycations on aglass substrate are schematically illustrated in FIG. 20 .

To facilitate covalent attachment to the glass substrate, polycationsbearing alkynyl moieties were prepared. These polycations could thenparticipate in click reactions with azide moieties introduced onto theglass substrate, thereby covalently bonding the polycation to the glasssubstrate. Polycations bearing alkynyl moieties were prepare using thesynthetic methodology outlined in Scheme 4 below.

Briefly, the cyclic bis-electrophile 1 (21 mg, 0.1 mmol, 1 equiv),silver nitrate (34 mg, 0.2 mmol, 2 equiv) and a bispyridinedinucleophile of interest (0.1 mmol, 1 equiv) were mixed in 0.5 mL DMSOand stirred overnight at room temperature under inert atmosphere. Aftercentrifugation, any solid material was filtered out and the resultingsolution was added to 15 mL CH₂Cl₂ or toluene to precipitate the desiredproduct. The material was isolated by filtration, washed with CH₂Cl₂ anddried to give a yellow sticky solid. The structure of polycationsbearing alkynyl moieties prepared using this methodology, along withpreliminary characterization data, are include

TABLE 7 Examples of polycations bearing alkynyl moieties prepared usingthe methodology outlined in Scheme 2 (but with varying nucleophilicmonomers. nucleophile Mn R_(h) ^(b) nucleophile Mn R_(h) ^(b) monomerPolymer n^(a) (Da) (nm) monomer Polymer n^(a) (Da) (nm)

3b 12 6200 77

3c 8 5800 —

3d 10 5500 35 3i 18 8800 151

3k 18 8400 —

3l 18 8600 —

One prepared, the polycations were covalently attached to the surface ofglass cover slips using the procedures detailed below. First, the glasscover slips were rendered amenable to click chemistry modification byinitial attachment of azide groups via standard siloxide coatingmethods. Briefly, 1 cm×1 cm silicon or glass chips or glass beads werecleaned with piranha solution for 30 mM, sonicated with deionized waterfor another 30 min, rinsed with methanol and dried with N₂. Theresulting clean substrate was immersed in a solution of (3-azidopropyl)triethoxysilane in toluene (20 mM) for 12 hours. The glass or siliconsubstrate was removed from the reaction mixture, washed with methanol,and dried.

Subsequently, the azide-functionalized slips were reacted with apolycation bearing alkyne groups in a Cu(I)-catalyzed azide-alkynecycloaddition (CuAAC) reaction. Briefly, the azide-functionalized glasswas immersed in a solution of the polycation of interest (1 mg/mL in80/20 water/t-BuOH mixture) containing copper sulfate (5% equivalentswith respect to alkyne), sodium ascorbate (10 mM) and THPTA ligand (1equivalent with respect to Cu) for 12 hours. After the reaction solutionwas away from the material, the surface was further cleaned by immersionin a 1 mM solution of cysteine to remove metal ions that may be bound tothe surface, followed by washing with methanol and drying.

The cytotoxicity of the polycations deposited on the glass cover slipswas then evaluated. Briefly, the bacteria were collected bycentrifugation at 4,000×g for 3 min at 4° C., washed with sterile PBS(pH 7.4) and finally suspended in PBS to get a final concentration of6×10⁶ cells mL⁻¹. The glass/silicon substrate (0.01-0.04 cm² in area)was added into the bacteria suspension and shaked for 0.5-4 hours. Then25 ul of suspension was spread onto a sterile Petri dishes covered witha layer of LB medium containing 0.8% agar (previously autoclaved, andcooled to 37° C.). After overnight incubation at 37° C., bacterialcolonies became visible and were counted and compared with the untreatedbacteria plate. The MIC was defined as the minimum concentration in thediluted series when the CFU number on the agar plate reached no morethan 10% of the control plate.

The results are shown in FIG. 17 . Polycation-functionalized glass coverslips, approximately 0.01 cm² in area, were found to abolish (E. coli)or significantly inhibit (P. aeruginosa) the ability of bacteria to growon agar plates after exposure. If dispersed in solution, the overallconcentration of the polycations would have been approximately 0.01-0.3μg/mL.).

As a control, azide-functionalized glass cover slips were similarlyreacted with a small quaternary alkyne(N,N,N-trimethylprop-2-yn-1-ammonium bromide). These control slips werenot effective at reducing bacterial growth, demonstrating that thatcytotoxicity resulted from the attached polycationic polymer.

To confirm that cytotoxicity did not result from leaching of thepolycation into the culture, the polycation-functionalized glass coverslip was vigorously washed before being immersed in the bacterialculture. The pre-washed polycation-functionalized glass cover slip stilldeactivated the bacterial culture, suggesting that leaching is not amajor component of cytotoxic activity.

Example 5. Covalent Attachment of Polycations to Polyvinyl Chloride(PVC) Substrates

Numerous conditions, including ventilator-associated pneumonia andcatheter-related urinary tract infections, are linked to the formationof biofilms and colonization of bacteria on medical tubing, which thenleads to a bacterial infection. These conditions are associated withhigh mortality rates, as well as increased hospital stay lengths andmedical care costs. Currently, using silver-coated tubing is the maineffort to minimize these infections in intubated patients, however, thisis not FDA approved for pediatric use.

To address these needs, the covalent linkage of anti-microbial agents toexisting medical tubing to reduce/eliminate the growth of pathogenicbacteria was investigated. Covidien Mallinckrodt cuffless endotrachealtubing (4.00 mm ID; PVC) was used as a substrate for proof of principlestudies. Anti-microbial agents were covalently attached to the surfaceof the medical tubing. The functionalized materials was then be testedfor bacterial adhesion using hospital-relevant strains: P. aeruginosa,S. aureus, E. coli. In this way, different modifications of thesubstrate could be screened in a time-efficient manner to developalternative strategies for minimizing biofilm and colonization ofbacteria and fungi on medical tubing. These strategies are schematicallyillustrated in FIG. 18 .

In a first example, the PVC of an example segment of medical tubing wasfunctionalized with example polycations using a CuAAC) reaction. In thefirst step, one or more PVC surfaces of the medical tubing (the interiorsurface, the exterior surface, or both the interior surface and theexterior surface) were derivatized with azide. Subsequently, the azideson the derivatized PVC were reacted with alkynes present in thepolycationic polymer in an azide-alkyne cycloaddition reaction tocovalently attach the polycationic polymer to the PVC surface.

Strategies used to immobilize the polycations described herein on a PVCsubstrate (in this case PVC tubing) are outlined in FIG. 21 .

To introduce azide moieties onto the surface of the PVC, aphase-transfer catalyst was used to facilitate the substitution reactionof chloride for azide on the solvent-exposed surface. Briefly, initialstudies were performed using commercial plain endotracheal tubing (ET)(Covidien Mallinckrodt Oral/Nasal Tracheal Tube Cuffless, 4.0 mm innerdiameter, 5.6 mm outer diameter) and catheter tubing (CT) (GentleCathIntermittent Urinary Catheter, Size 14 Male Nelaton 16″) purchased fromthe indicated vendors. Test reactions were performed as follows.

Small pieces of clear, cut tubing (approximately 0.5×0.5 cm) were placedin 4 mL of an aqueous solution of 450 mM NaN₃ and 9 mM phase transfercatalyst in sealed vials. Each mixture was heated to 60° C., 80° C., or100° C. for periods from 12 to 48 hours while being shaken vigorously onan orbital shaker. The samples were then placed in water and sonicatedfor 1 hour to rinse, changing the water at 30 minutes, and then dried inan oven at 60° C. for 4-8 hours. Using this methodology, the amount ofazide introduced onto the surface of the PVC tubing could be readilycontrolled without leaching plasticizers or changing the bulk mechanicalproperties of the PVC.

Subsequently, anti-bacterial and anti-fungal molecules were covalentlyattached to the PVC. In these proof of principle studies, polycationicpolymers based on cationic centers formed by the reaction of a9-thia/aza/selenabicyclo[3.3.1]nonyl electrophile with a nucleophile.Each bond-forming event generates a positive charge. FIG. 19 illustratesexamples of polycationic polymers that can be prepared through thereaction of a 9-thia/aza/selenabicyclo[3.3.1]nonyl electrophile with anucleophile. Type I materials are prepared through the condensation of9-thia/aza/selenabicyclo[3.3.1]nonyl dielectrophiles withpolynucleophiles (e.g., dinucleophiles). Each bond-forming event in thiscondensation polymerization process generates a positively chargedcenter within the polymer backbone. Type II materials contain cationicsidechains. Type II materials can be formed, for example, bypolymerization of monomers (e.g., acrylate monomers) containing pendant9-azabicyclo[3.3.1]nonyl electrophiles. Following polymerization, thependant 9-azabicyclo[3.3.1]nonyl electrophiles can be reacted withnucleophiles to introduce positively charged centers within the polymersidechains. If desired, conventional anti-microbial compounds (e.g.,antibiotics) can be used in place of (or in addition to) thesepolycationic polymers.

In a second example, the PVC was derivatized with alternative functionalgroups (other than azides) that could subsequently be reacted withanti-bacterial and/or anti-fungal molecules. Other moieties that canserve as reactive functional groups to facilitate surfacefunctionalization include, for example, thiolate, thiocyanate (SCN⁻),hydroxide, iodide, and nitrile groups. In a second example, the PVC wascyano-functionalized. Briefly, small pieces of clear, cut tubing(approximately 0.5×0.5 cm) were placed in 4 mL of an aqueous solution of450 mM NaCN and 9 mM phase transfer catalyst in sealed vials. Eachmixture was heated to 60° C., 80° C., or 100° C. for periods from 12 to48 hours while being shaken vigorously on an orbital shaker. The sampleswere then placed in water and sonicated for 1 hour to rinse, changingthe water at 30 minutes, and then dried in an oven at 60° C. for 4-8hours.

Subsequently, the nitrile groups on the derivatized PVC were reactedsodium azide to produce 1H-tetrazoles. Briefly, the cyanated PVC sampleswere immersed in an aqueous solution of sodium azide (485 mM) zincbromide (440 mM) at 100° C. for 72 h to form 5-substituted 1H-tetrazoleson the material surface. These surfaces were then alkylated with anantimicrobial compound or with a compound containing an azide or alkynegroup to which subsequent connection can be made by azide-alkynecycloaddition. By way of example, the samples were further immersed in asolution of 360 mM potassium carbonate and 48 mM of the electrophile2,3,4,5,6-pentafluorobenzyl bromide, and shaken at 80° C. for 12 hours.Alternatively, nitrile groups on the derivatized PVC could be reactedwith polycationic polymers bearing hydrazone moieties (which condensewith the nitrile groups to form 1,2,4-triazoles).

The compositions, devices, systems, and methods of the appended claimsare not limited in scope by the specific compositions, devices, systems,and methods described herein, which are intended as illustrations of afew aspects of the claims. Any compositions, devices, systems, andmethods that are functionally equivalent are intended to fall within thescope of the claims. Various modifications of the compositions, devices,systems, and methods in addition to those shown and described herein areintended to fall within the scope of the appended claims. Further, whileonly certain representative compositions, devices, systems, and methodsteps disclosed herein are specifically described, other combinations ofthe compositions, devices, systems, and method steps also are intendedto fall within the scope of the appended claims, even if notspecifically recited. Thus, a combination of steps, elements,components, or constituents may be explicitly mentioned herein or less,however, other combinations of steps, elements, components, andconstituents are included, even though not explicitly stated.

The term “comprising” and variations thereof as used herein is usedsynonymously with the term “including” and variations thereof and areopen, non-limiting terms. Although the terms “comprising” and“including” have been used herein to describe various embodiments, theterms “consisting essentially of” and “consisting of” can be used inplace of “comprising” and “including” to provide for more specificembodiments of the invention and are also disclosed. Other than wherenoted, all numbers expressing geometries, dimensions, and so forth usedin the specification and claims are to be understood at the very least,and not as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, to be construed in light of thenumber of significant digits and ordinary rounding approaches.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

What is claimed is:
 1. A process for rendering a polyvinyl chloride surface antimicrobial, the process comprising: (a) contacting the polyvinyl chloride surface with a cyanation reagent in the presence of a phase transfer catalyst to form a cyano-substituted polyvinyl chloride surface; and (b) covalently bonding an antimicrobial agent to the cyano-substituted polyvinyl chloride surface.
 2. The process of claim 1, wherein the antimicrobial agent comprises a hydrazone moiety or an azide moiety, and wherein step (b) comprises contacting the cyano-substituted polyvinyl chloride surface with the antimicrobial agent under conditions effective to covalently bond the antimicrobial agent to the polyvinyl chloride surface.
 3. The process of claim 1, wherein step (b) comprises: (i) converting cyano groups in the cyano-substituted polyvinyl chloride surface to 1H-tetrazole moieties, thereby forming a 1H-tetrazole-substituted polyvinyl chloride surface; and (ii) contacting the 1H-tetrazole-substituted polyvinyl chloride surface with an antimicrobial agent comprising an electrophilic moiety under conditions effective to covalently bond the antimicrobial agent to the polyvinyl chloride surface.
 4. The process of claim 3, wherein step (i) comprises contacting the cyano-substituted polyvinyl chloride surface with an azide salt in the presence of a catalyst.
 5. The process of claim 1, wherein step (b) comprises: (i) converting cyano groups in the cyano-substituted polyvinyl chloride surface to 1H-tetrazole moieties, thereby forming a 1H-tetrazole-substituted polyvinyl chloride surface; (ii) contacting the 1H-tetrazole-substituted polyvinyl chloride surface with an electrophile comprising an azide group to form an azide-substituted polyvinyl chloride surface; and (iii) contacting the azide-substituted polyvinyl chloride with an antimicrobial agent comprising an alkyne moiety under conditions effective to covalently bond the antimicrobial agent to the polyvinyl chloride surface.
 6. The process of claim 5, wherein the electrophile comprising the azide group is chosen from a benzylic halide comprising an azide group, an allylic halide comprising an azide group, and a propargylic halide comprising an azide group.
 7. The process of claim 5, wherein the alkyne moiety is activated by ring strain, electron withdrawing groups, or a combination thereof.
 8. The process of claim 5, wherein step (iii) comprises contacting the azide-substituted polyvinyl chloride surface with the antimicrobial agent comprising the alkyne moiety in the presence of a Cu(I) catalyst.
 9. The process of claim 1, wherein step (b) comprises: (i) converting cyano groups in the cyano-substituted polyvinyl chloride surface to 1H-tetrazole moieties, thereby forming a 1H-tetrazole-substituted polyvinyl chloride surface; (ii) contacting the 1H-tetrazole-substituted polyvinyl chloride surface with an electrophile comprising an alkynyl group to form an alkyne-substituted polyvinyl chloride surface; and (iii) contacting the alkyne-substituted polyvinyl chloride surface with an antimicrobial agent comprising an azide moiety under conditions effective to covalently bond the antimicrobial agent to the polyvinyl chloride surface.
 10. The process of claim 9, wherein the electrophile comprising the alkynyl group is chosen from a benzylic halide comprising an alkynyl group, an allylic halide comprising an alkynyl group, and a propargylic halide comprising an alkynyl group.
 11. The process of claim 9, wherein the alkynyl group is activated by ring strain, electron withdrawing groups, or a combination thereof.
 12. The process of claim 9, wherein step (iii) comprises contacting the alkyne-substituted polyvinyl chloride with the antimicrobial agent comprising the azide moiety in the presence of a Cu(I) catalyst.
 13. The process of claim 1, wherein step (a) comprises flowing an aqueous solution comprising the cyanation reagent and the phase transfer catalyst across the polyvinyl chloride surface.
 14. The process of claim 1, wherein step (a) comprises contacting the polyvinyl chloride surface with the cyanation reagent and the phase transfer catalyst for 3 hours or less.
 15. The process of claim 1, wherein the polyvinyl chloride surface comprises a surface of a medical article.
 16. The process of claim 15, wherein the medical article comprises tubing.
 17. The process of claim 16, wherein the surface of the medical article comprises an interior surface of the tubing, an exterior surface of the tubing, or both an interior surface of the tubing and an exterior surface of the tubing.
 18. The process of claim 1, wherein the antimicrobial agent comprises a polycationic polymer.
 19. The process of claim 18, wherein the polycationic polymer comprises a polymer defined by Formula I below

wherein Z represents, individually for each occurrence, S, Se, or NR³; A represents, individually for each occurrence, a heterocyclic ring comprising a cationic nitrogen center; X represents, individually for each occurrence, an anion; Y is absent, or represents, individually for each occurrence, a linking group; R¹ is absent, or represents, individually for each occurrence, halogen, hydroxy, amino, cyano, azido, hydrazone, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl, cycloalkyl alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, dialkylamino, alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, peptidyl, polyamino, or polyalkyleneoxy; R² is absent, or represents, individually for each occurrence, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl, cycloalkyl alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl, peptidyl, polyamino, or polyalkyleneoxy; R³ represents, individually for each occurrence, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl, cycloalkyl alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl, peptidyl, polyamino, or polyalkyleneoxy; and n is an integer from 2 to
 400. 20. The process of claim 19, A is chosen from pyridine, imidazole, pyrazole, triazole, tetrazole, oxazole, isoxazole, furazan, isothioazole, and thiazole.
 21. The process of claim 19, wherein Z is S.
 22. The process of claim 19, wherein Z is NR³.
 23. The process of claim 19, wherein R¹, R², R³, or a combination thereof comprise a cationic moiety.
 24. The process of claim 19, wherein Y is chosen from one of the following:

where m is an integer from 1 to
 12. 25. The process of claim 19, where X is chosen from chloride, bromide, iodide, nitrate, sulfate, triflate, borate, and phosphate.
 26. The process of claim 19, wherein n is from 10 to
 50. 27. The process of claim 19, wherein the polycationic polymer comprises a polymer defined by Formula IA below

wherein Z, X, Y, R¹, R², R³, and n are as defined above with respect to Formula I.
 28. The process of claim 18, wherein the polycationic polymer comprises a recurring unit defined by Formula V below

wherein R⁴ represents H or methyl; L is absent or represents a linking group; A represents, individually for each occurrence, a heterocyclic ring comprising a cationic nitrogen center; R⁵ is absent, or represents, individually for each occurrence, halogen, hydroxy, amino, cyano, azido, hydrazone, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, alkylaryl, alkylheteroaryl, cycloalkyl alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, dialkylamino, alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, peptidyl, polyamino, or polyalkyleneoxy; and m is an integer from 0 to
 5. 29. The process of claim 28, wherein the polycationic polymer further comprises a recurring unit derived from the polymerization of one or more ethylenically-unsaturated monomers.
 30. The process of claim 29, wherein the one or more ethylenically-unsaturated monomers are chosen from the group consisting of styrene, butadiene, meth(acrylate) monomers, vinyl acetate, vinyl ester monomers and combinations thereof.
 31. The process of claim 1, wherein the phase transfer catalyst comprises a quaternary ammonium salt.
 32. The process of claim 1, wherein the cyanation reagent comprises NaCN, KCN, or a combination thereof. 